V. Van Hoeck

The effect of nutritionally induced hyperlipidaemia on in vitro bovine embryo quality

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 RT-PCR. 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 micromol/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.

Intrafollicular conditions as a major link between maternal metabolism and oocyte quality: a focus on dairy cow fertility

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 oocytes 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.

Interaction between differential gene expression profile and phenotype in bovine blastocysts originating from oocytes exposed to elevated non-esterified fatty acid concentrations

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.

Negative energy balance and metabolic stress in relation to oocyte and embryo quality: an update on possible pathways reducing fertility in dairy cows

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 microenvironment 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.