P.E.J. Bols

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.

Maternal metabolic stress may affect oviduct gatekeeper function

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.

The effect of a negative energy balance status on β-carotene availability in serum and follicular fluid of nonlactating dairy cows.

Maternal metabolic pressure due to a cows negative energy balance (NEB) has a negative effect on oocyte quality as a result of increased oxidative stress. In this study, we hypothesized that a NEB status may negatively affect the availability of β-carotene (bC, an antioxidant) in the micro-environment of the oocyte or follicular fluid (FF) and that daily bC supplementation can increase bC availability. We aimed to (1) determine the effect of a nutritionally induced NEB on bC concentrations in serum and FF as well as on the presence of bC metabolites, oxidative stress levels, and follicular growth in a nonlactating dairy cow model, and (2) investigate how this effect could be altered by dietary bC supplementation. Six multiparous nonlactating Holstein Friesian cows were subjected to 4 consecutive dietary treatments, 28 d each: (1) 1.2 × maintenance (M) or positive energy balance (PEB) without bC supplement (PEB-bC), (2) 1.2 × M with daily supplement of 2,000mg of bC comparable to the level of bC intake at grazing (PEB+bC), (3) 0.6 × M with 2,000mg of bC (NEB+bC), and (4) 0.6 × M (NEB-bC). At the end of each treatment, estrous cycles were synchronized and blood and FF of the largest follicle were sampled and analyzed for bC, retinol, α-tocopherol, free fatty acids, estradiol, and progesterone. Serum cholesterol, triglycerides, urea, insulin growth factor 1, growth hormone, total antioxidant status (TAS), and red blood cell glutathione (GSH) concentrations were determined as well. All cows lost body weight during both energy restriction periods and showed increased serum free fatty acid concentrations, illustrating a NEB. A dietary induced NEB reduced FF bC, but not plasma bC or plasma and FF retinol concentrations. However, bC and retinol concentrations drastically increased in both fluid compartments after bC supplementation. Follicular diameter was increased in supplemented PEB cows. Energy restriction reduced the TAS and red blood cell GSH, whereas daily bC supplementation could restore GSH concentrations, but not the TAS, to levels present in healthy PEB cows. In conclusion, daily bC supplementation can substantially improve bC and retinol availability in the oocytes micro-environment, irrespective of the energy balance, which may affect follicular development and oocyte quality in the presence of maternal metabolic stress. This knowledge can be of importance to optimize nutritional strategies in the dairy industry to feed for optimal oocyte quality and fertility.

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.

Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species.

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

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

PurposeIndividual 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.MethodsBovine 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).ResultsNo 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.ConclusionsOur 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.