Effects of vitrification on the viability of alginate encapsulated isolated bovine pre-antral follicles.

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 using mesh cups (3DVIT), (2) culture in 2% alginate beads (3DCUL), (3) culture in 96-well plates (2D system) and vitrification using High Security Vitrification straws® (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 3DVIT showed 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 impaired morphology, we consider it advantageous to optimize the protocol for the vitrification of embedded follicles to increase survival and maintain morphology after vitrification.

Peri-ovulatory endocrine regulation of the prostanoid pathways in the bovine uterus at early dioestrus.

We hypothesised that different endocrine profiles associated with pre-ovulatory follicle (POF) size would impact on uterine prostanoid pathways and thereby modulate the histotroph composition. Beef cows (n=15 per group) were hormonally manipulated to have small (SF-SCL group) or large (LF-LCL group) pre-ovulatory follicles (POF) and corpora lutea (CL). Seven days after induction of ovulation, animals were slaughtered and uterine tissues and flushings were collected for quantification of prostanoids. The POF and CL size and the circulating progesterone concentrations at Day 7 were greater (P<0.05) in the LF-LCL cows than in the SF-SCL group, as expected. The abundance of 5 out of 19 genes involved in prostanoid regulation was different between groups. Transcript abundance of prostaglandin F2α, E2 and I2 synthases was upregulated (P<0.05) and phospholipase A2 was downregulated (P<0.05) in endometrium of the LF-LCL group. No difference (P>0.1) in prostanoid concentrations in the endometrium or in uterine flushings was detected between groups. However, prostaglandin F2α and E2 concentrations in the uterine flushings were positively correlated with the abundance of transcripts for prostaglandin endoperoxide synthase 2 (0.779 and 0.865, respectively; P<0.002). We conclude that endometrial gene expression related to prostanoid synthesis is modulated by the peri-ovulatory endocrine profile associated with POF size, but at early dioestrus differences in transcript abundance were not reflected in changes in prostanoid concentrations in the uterine tissue and fluid.

Effect of nutritionally induced hyperlipidaemia on in vitro bovine embryo quality depends on the type of major fatty acid in the diet.

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.

Nutrition and maternal metabolic health in relation to oocyte and embryo quality: critical views on what we learned from the dairy cow model.

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.

Spatio-specific regulation of endocrine-responsive gene transcription by periovulatory endocrine profiles in the bovine reproductive tract.

In cattle, pro-oestrous oestradiol and dioestrous progesterone concentrations modulate endometrial gene expression and fertility. The aim was to compare the effects of different periovulatory endocrine profiles on the expression of progesterone receptor (PGR), oestrogen receptor 2 (ESR2), oxytocin receptor (OXTR), member C4 of aldo-keto reductase family 1 (AKR1C4), lipoprotein lipase (LPL), solute carrier family 2, member 1 (SLC2A1) and serpin peptidase inhibitor, clade A member 14 (SERPINA14): (1) between uterine horns ipsi- and contralateral to the corpus luteum (CL), (2) between regions of the ipsilateral horn and (3) in the vagina. Endometrium and vagina tissue samples were collected from cows that ovulated a larger (large follicle-large CL, LF-LCL; n=6) or smaller follicle (small follicle-small CL, SF-SCL; n=6) 7 days after oestrus. Cows in the LF-LCL group had a greater abundance of transcripts encoding ESR2, AKR1C4, LPL, SLC2A1 and SERPINA14, but a reduced expression of PGR and OXTR in the endometrium versus the SF-SCL group (PPGR and OXTR was greater in the contralateral compared with the ipsilateral horn (PPGR, ESR2, LPL, SLC2A1 and SERPINA14 (P<0.05). Different periovulatory endocrine profiles, i.e. LF-LCL or SF-SCL, did not influence gene expression in the vagina and had no interaction with inter- or intra-uterine horn gene expression. In conclusion, inter- and intra-uterine horn variations in gene expression indicate that the expression of specific genes in the bovine reproductive tract is location dependent. However, spatial distribution of transcripts was not influenced by distinct periovulatory sex-steroid environments.

Untargeted UHPLC-TOF/MS Lipidomic Analysis for the Investigation of Egg Yolks after Xylanase Supplementation of the Diet of Laying Hens.

Xylanase supplementation of diets is used to enhance nutrient digestibility in monogastrics which lack necessary enzymes for non-starch polysaccharide degradation. The effects of enzymatic treatment in the nutritional value of the feed are typically not comprehensively studied. Though the fundamental effects of xylanase on performance are well studied, limited data is available on the complex interactions between xylanase supplementation and hen physiology; therefore, the aim of this study was to develop a new, simple UPLC-TOF/MS lipidomics method for the analysis of hen egg yolks after supplementation with different amounts of xylanase. Sample preparation for the extraction of lipids was optimized and different sample preparation modes and solvent mixtures were tested. Optimal results for the extraction of total lipids were obtained by using the solvent mixture MTBE: MeOH (5:1, v/v). Multivariate statistical analysis of the signals of hundreds of lipids in positive and negative ionisation modes highlighted differences in several egg yolk lipid species-classes. Four lipid species-classes, phosphatidylcholines (PC and PC O), phosphatidylethanolamines (PE and PE O), phosphatidylinositols (PI), and fatty acids (FA), were among those contributing to the separation of the experimental groups (control-treated) in negative ionisation mode. In positive ionisation mode, principal beneficial lipid compounds such as phosphatidylcholines (PC and PC O), phosphatidylethanolamines (PE and PE O), triacylglycerols (TG), diacylglycerols (DG), and ceramides (Cer) were found to be increased in treated groups. Overall, supplementation of laying hens' diets with xylanase significantly changed the lipid profile of egg yolks compared to the control diet. The association between the lipid profiles of egg yolks and hens' diets, as well as the underlying mechanisms, require further investigation. These findings are of practical significance for the food industry.

Xylanase Impact beyond Performance: Effects on Gut Structure, Faecal Volatile Fatty Acid Content and Ammonia Emissions in Weaned Piglets Fed Diets Containing Fibrous Ingredients.

The addition of xylanase to piglet diets is known to improve performance and nutrient digestibility. The present study aimed to assess the impact of new xylanase on the growth performance, nutrient digestibility, and gut function of weaned piglets. A total of 144 pigs, weaned at 28 days (7.48 kg initial body weight, IBW), were assigned to 36 pens and 9 pens per treatment. Dietary treatments were a basal complex control diet, and the basal diet supplemented with 45,000, 90,000 and 135,000 U/kg xylanase. Performance was measured at days 0, 14 and 35. At day 35, samples were collected for assessment of intestinal histology, and volatile fatty acid and ammonia concentrations. After two weeks post-weaning, additional 12 piglets (11.34 kg IBW) were placed in metabolic crates for assessment of apparent total tract nutrient digestibility using a dietary marker. The addition of xylanase at 90,000 and 135,000 U/kg significantly improved average daily gain (333.6 g/day control, 364.86 g/day, 90,000 U/kg, 405.89 g/day, 135,000 U/kg, p < 0.05), G:F (0.557 control, 0.612 90,000 U/kg, 0.692 135,000 U/kg, p < 0.05), and reduced diarrhoea. This was driven improved nutrient digestibility and villus height in the jejunum (372.87 µm control, 432.53 µm 45,000 U/kg, 465.80 µm 90,000 U/kg, 491.28 µm 135,000 U/kg, p < 0.05). Xylanase supplementation also linearly increased faecal butyrate levels and had a quadratic relationship with propionate concentrations. 135,000 U/kg xylanase also reduced ammonia emissions. In conclusion, dietary supplementation with xylanase improved growth performance and feed efficiency in weaning piglets, likely driven by improvements to gut structure and function.

Xylanase Supplementation in Wheat-Based Diets of Laying Hens Affects the Egg Yolk Color, Carotenoid and Fatty Acid Profiles.

Wheat is rich in non-starch polysaccharides (NSP) and their degradation in poultry diets is promoted by exogenous carbohydrases. The objective here was to evaluate the effect of adding an intrinsically thermostable xylanase on wheat-based diets for laying hens in yolk color, carotenoid and fatty acid profiles of eggs. A total of 128 laying hens were used for 12 weeks. They were randomly allocated to four dietary treatments with different levels of xylanase: T1: control (no xylanase), T2: 30,000 U/g, T3: 45,000 U/g and T4: 90,000 U/g, with 32 birds, 16 replicates per treatment (2 birds/replicate). At the end of the experimental period, egg yolk color index, redness (a*) and yellowness (b*) of egg yolks were found significantly higher in all the enzyme supplemented diet groups (T2, T3, T4) compared with the control (T1). Canthaxanthin levels were significantly higher in T3 than T1 (p < 0.05). Total n-3, n-6 and total polyunsaturated fatty acids (FAs) were significantly higher in T4 compared with the control (p < 0.01), while the reverse trend was evidenced for monounsaturated FAs. Additionally, total n-3 FAs were higher in the T2 than T1 (p < 0.005). Overall, the results showed that exogenous xylanase enzyme supplementation in wheat-based diets for laying hens contribute to maintaining egg yolk color. Overall, exogenous xylanase enzyme supplemented at all levels in wheat-based laying hens' diets improved egg yolk color compared to the control diet. The enzyme supplemented at the higher level (90,000 U/g) improved polyunsaturated and reduced monounsaturated egg yolk fatty acid content.

Endometrial receptivity in cattle: the mutual reprogramming paradigm.

Prior to implantation in cattle, the mucous medium contained in the uterine lumen serves as a working interface for molecular exchange and signaling between the lining endometrium and the embryo. The composition of this luminal fluid changes temporally according to the secretory and reabsorptive activities of the uterus and the embryo, which are under complex regulation. Via this interface, both the embryo and the endometrium reprogram each other's functions to support pregnancy continuation beyond the pre-implantation period. More specifically, the embryo receives elongation signals and the uterus receives anti-luteolytic stimuli. Here, characteristics of the luminal compartment as well as the regulation of its composition to determine the pregnancy outcome will be discussed.

Xylanase impact beyond performance: A microbiome approach in laying hens.

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 (P<0.05). Egg quality parameters were significantly improved in the experiment in response to the xylanase addition. For example, during the last 28 days of the trial, birds receiving the 45,000 U/kg and the 90,000 U/kg treatments exhibited an increase in Haugh units and albumin heights (P<0.05). Compared with the control, the ATTD of organic matter and crude protein were drastically improved in the 45,000 U/kg treatment group (P<0.05). Furthermore, gross energy and the ATTD of crude fat were improved significantly for birds fed 90,000 U/kg group compared to the control. Importantly, 16S rRNA gene analysis revealed that xylanase at 45,000 U/kg dosage can exert a change in the cecal microbiome. A significant increase in beneficial bacteria (Bacilli 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.

Chromium propionate improves performance and carcass traits in broilers.

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 < 0.05), feed efficiency (P < 0.05) and body weight gain (P < 0.0001). Furthermore, Cr propionate supplementation improved (P < 0.0001) all carcass characteristics. Interestingly, with increased Cr dosage, carcass yield, dressing percentage and breast meat yield increased linearly (P < 0.0001). The second study reveals that the feed intake in the control group was significantly higher compared to both Cr propionate supplemented groups (T1 & T2). Furthermore, the Cr propionate supplemented T2 group displayed a significantly lower FCR than the control and T1 group (P = 0.027). Finally, Cr propionate supplementation increased the dressing percentage compared to control birds (P < 0.0001). In the third experiment, Cr propionate supplementation (T1 & T2) increased final body weight and decreased FCR compared with the control treatment. These effects were highly significant (P < 0.0001) throughout all feeding phases of the trial. Cr propionate supplementation also increased (P < 0.0001) carcass yield, dressing percentage, breast meat yield, leg and thigh weights compared with the control treatment. In conclusion, growth performance, feed conversion, carcass yield, breast and leg meats of broiler birds can be significantly improved by dietary inclusion of Cr propionate. Cr propionate can be supplemented to broiler birds from 1 d old of age at a level that provides 200 or 400 µg/kg organic Cr and can increase the efficiency of broiler production.

Evidence of endometrial amino acid metabolism and transport modulation by peri-ovulatory endocrine profiles driving uterine receptivity.

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 α-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.

Non-esterified fatty acids in early luteal bovine oviduct fluid mirror plasma concentrations: An ex vivo approach.

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±0.14mmol/L, they didn't 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.

Oviductal transcriptional profiling of a bovine fertility model by next-generation sequencing.

In cattle, the oviduct plays a fundamental role in the reproductive process. Oviductal functions are controlled by the ovarian sex steroids: estradiol and progesterone. Here, we tested the hypothesis that the exposure to contrasting sex steroid milieus differentially impacts the oviductal transcriptional profile. We manipulated growth of the pre-ovulatory follicle to obtain cows that ovulated a larger (LF group) or a smaller (SF group) follicle. The LF group presented greater proestrus/estrus concentrations of estradiol and metaestrus concentrations of progesterone (Gonella-Diaza et al. 2015 [1], Mesquita et al. 2014 [2]). Also, the LF group was associated with greater fertility in timed-artificial insemination programs (Pugliesi et al. 2016 [3]). Cows were slaughtered on day 4 of the estrous cycle and total RNA was extracted from ampulla and isthmus fragments and analyzed by RNAseq. The resulting reads were mapped to the bovine genome (Bos taurus UMD 3.1, NCBI). The differential expression analyses revealed that 325 and 367 genes in ampulla and 274 and 316 genes in the isthmus were up-regulated and down-regulated in LF samples, respectively. To validate the RNAseq results, transcript abundance of 23 genes was assessed by qPCR and expression patterns were consistent between the two techniques. A functional enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) software. Processes enriched in the LF group included tissue morphology changes (extracellular matrix remodeling), cellular changes (proliferation), and secretion changes (growth factors, ions and metal transporters). An overview of the gene expression data was deposited in the NCBI's Gene Expression Omnibus (GEO) and is accessible through the accession number GSE65681. In conclusion, differences in the peri-ovulatory sex steroid milieu modify the oviductal gene expression profiles. Such differences may be associated with the greater fertility of the LF cows. This dataset is useful for further investigations of the oviductal biology and the impact of sex-steroid on the female reproductive tract.

Gene expression profiling by high throughput sequencing to determine signatures for the bovine receptive uterus at early gestation.

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.

The transcriptome signature of the receptive bovine uterus determined at early gestation.

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.

Size of the Ovulatory Follicle Dictates Spatial Differences in the Oviductal Transcriptome in Cattle.

In cattle, molecular control of oviduct receptivity to the embryo is poorly understood. Here, we used a bovine model for receptivity based on size of the pre-ovulatory follicle to compare oviductal global and candidate gene transcript abundance on day 4 of the estrous cycle. Growth of the pre-ovulatory follicle (POF) of Nelore (Bos indicus) cows was manipulated to produce two groups: large POF large corpus luteum (CL) group (LF-LCL; greater receptivity) and small POF-small CL group (SF-SCL). Oviductal samples were collected four days after GnRH-induced ovulation. Ampulla and isthmus transcriptome was obtained by RNA-seq, regional gene expression was assessed by qPCR, and PGR and ERa protein distribution was evaluated by immunohistochemistry. There was a greater abundance of PGR and ERa in the oviduct of LF-LCL animals thus indicating a greater availability of receptors and possibly sex steroids stimulated signaling in both regions. Transcriptomic profiles indicated a series of genes associated with functional characteristics of the oviduct that are regulated by the periovulatory sex steroid milieu and that potentially affect oviductal receptivity and early embryo development. They include tissue morphology changes (extra cellular matrix remodeling), cellular changes (proliferation), and secretion changes (growth factors, ions and metal transporters), and were enriched for the genes with increased expression in the LF-LCL group. In conclusion, differences in the periovulatory sex steroid milieu lead to different oviductal gene expression profiles that could modify the oviductal environment to affect embryo survival and development.

Reduced oocyte and embryo quality in response to elevated non-esterified fatty acid concentrations: a possible pathway to subfertility?

Reproductive performance is increasingly considered as a 'barometer' of general well-being of the mother. A normal maternal 'metabolic health' status is essential to safeguard successful ovulation, conception and further embryo development. When alterations in serum metabolites are reflected in the oocyte and embryonic micro-environment, these metabolic changes can affect follicle health, oocyte development and even subsequent embryo physiology. The search continues for signals that may be critically affecting the early developmental stages in life. Years of expertise in animal in vitro embryo culture models contribute to the awareness on the influence of elevated non-esterified fatty acid (NEFA) concentrations on follicle cells, oocyte and embryo quality. High NEFA concentrations in the blood are known to alter the follicular micro-environment. The latter alterations in NEFA concentrations have been associated with a disappointing fertility outcome through disabled ovarian cell function and reduced oocyte's developmental competence. Even more, elevated NEFA concentrations during bovine oocyte maturation influence the subsequent embryo characteristics. This review provides a cross-species overview on the consequences of elevated NEFA concentrations, originating from maternal lipolytic conditions, on female fertility, with particular focus on the early stages in life. Thereby, we will describe to what extent elevated serum NEFA concentrations are a potential threat around the period of conception.

Elevated non-esterified fatty acid concentrations during in vitro murine follicle growth alter follicular physiology and reduce oocyte developmental competence.

OBJECTIVE: To study how long-term elevated non-esterified fatty acid (NEFA) concentrations, typical in metabolic disorders such as obesity or type 2 diabetes, affect murine follicular development, follicle quality, and subsequent oocyte developmental competence in vitro. DESIGN: Experimental study. SETTING: In vitro culture setting. ANIMAL(S): Female and male 13-day old, B6CBAF1 mice of proven fertility were sacrificed for harvesting ovaries and epididymal sperm, respectively. INTERVENTION(S): Early secondary murine follicles were cultured in vitro in the presence of NEFAs until the antral stage (12 days). Treatments consisted of one or a mixture of NEFAs (stearic acid [SA], palmitic acid [PA], oleic acid [OA]) in physiological (basal) or pathological (high SA, high OA, high NEFA) concentrations. MAIN OUTCOME MEASURE(S): Follicular development; follicle and oocyte diameters; secretion of progesterone, estradiol, and inhibin B; and luteinized granulosa cell gene expression patterns were investigated. Oocytes from NEFA-exposed follicles were fertilized in vitro, and presumptive zygotes were cultured until the blastocyst stage. RESULT(S): Exposure to high SA reduced follicle diameters and day-12 antrum formation. Elevated NEFA concentrations changed luteinized granulosa cell messenger-ribonucleic acid abundance of genes related to energy/fatty acid/steroid metabolism, apoptosis, and oxidative stress. High NEFA and high SA treatments increased progesterone synthesis, compared with high OA follicles. Oocyte developmental competence was substantially reduced in oocytes retrieved from high OA-, high SA-, and high NEFA-exposed follicles compared with basal-treated follicles. CONCLUSION(S): This study showed, for the first time, that lipolysis-linked, elevated NEFA concentrations can potentially impair fertility, by altering follicular physiology and reducing oocyte developmental competence.

Elevated non-esterified fatty acid concentrations during bovine oocyte maturation compromise early embryo physiology.

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.