The molecular events associated with regulation of milk fat synthesis in the bovine mammary gland remain largely unknown. Our objective was to study mammary tissue mRNA expression via quantitative PCR of 45 genes associated with lipid synthesis (triacylglycerol and phospholipids) and secretion from the late pre-partum/non-lactating period through the end of subsequent lactation. mRNA expression was coupled with milk fatty acid (FA) composition and calculated indexes of FA desaturation and de novo synthesis by the mammary gland. Marked up-regulation and/or % relative mRNA abundance during lactation were observed for genes associated with mammary FA uptake from blood (LPL, CD36), intracellular FA trafficking (FABP3), long-chain (ACSL1) and short-chain (ACSS2) intracellular FA activation, de novo FA synthesis (ACACA, FASN), desaturation (SCD, FADS1), triacylglycerol synthesis (AGPAT6, GPAM, LPIN1), lipid droplet formation (BTN1A1, XDH), ketone body utilization (BDH1), and transcription regulation (INSIG1, PPARG, PPARGC1A). Change in SREBF1 mRNA expression during lactation, thought to be central for milk fat synthesis regulation, was ≤2-fold in magnitude, while expression of INSIG1, which negatively regulates SREBP activation, was >12-fold and had a parallel pattern of expression to PPARGC1A. Genes involved in phospholipid synthesis had moderate up-regulation in expression and % relative mRNA abundance. The mRNA abundance and up-regulation in expression of ABCG2 during lactation was markedly high, suggesting a biological role of this gene in milk synthesis/secretion. Weak correlations were observed between both milk FA composition and desaturase indexes (i.e., apparent SCD activity) with mRNA expression pattern of genes measured. A network of genes participates in coordinating milk fat synthesis and secretion. Results challenge the proposal that SREBF1 is central for milk fat synthesis regulation and highlight a pivotal role for a concerted action among PPARG, PPARGC1A, and INSIG1. Expression of SCD, the most abundant gene measured, appears to be key during milk fat synthesis. The lack of correlation between gene expression and calculated desaturase indexes does not support their use to infer mRNA expression or enzyme activity (e.g., SCD). Longitudinal mRNA expression allowed development of transcriptional regulation networks and an updated model of milk fat synthesis regulation.

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Gene networks driving bovine milk fat synthesis during the lactation cycle

Influence of negative energy balance on cyclicity and fertility in the high producing dairy cow.

The effect of nutritional management of the dairy cow on reproductive efficiency.

Metabolism and ATP levels within the oocyte and adjacent cumulus cells are associated with quality of oocyte and optimal development of a healthy embryo. Lipid metabolism provides a potent source of energy and its importance during oocyte maturation is being increasingly recognised. The triglyceride and fatty acid composition of ovarian follicular fluid has been characterised for many species and is influenced by nutritional status (i.e. dietary fat, fasting, obesity and season) as well as lactation in cows. Lipid in oocytes is a primarily triglyceride of specific fatty acids which differ by species, stored in distinct droplet organelles that re-localise during oocyte maturation. The presence of lipids, particularly saturated vs unsaturated fatty acids, in in vitro maturation systems affects oocyte lipid content as well as developmental competence. Triglycerides are metabolised by lipases that have been localised to cumulus cells as well as oocytes. Fatty acids generated by lipolysis are further metabolised by β-oxidation in mitochondria for the production of ATP. β-oxidation is induced in cumulus-oocyte complexes (COCs) by the LH surge, and pharmacological inhibition of β-oxidation impairs oocyte maturation and embryo development. Promoting β-oxidation with l-carnitine improves embryo development in many species. Thus, fatty acid metabolism in the mammalian COC is regulated by maternal physiological and in vitro environmental conditions; and is important for oocyte developmental competence.

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Lipids and oocyte developmental competence: the role of fatty acids and β-oxidation.

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.

Elevated Non-Esterified Fatty Acid Concentrations during Bovine Oocyte Maturation Compromise Early Embryo Physiology

In this study concentration and composition of non-esterified fatty acids (NEFA) in follicular fluid (FF) of high-yielding dairy cows were determined during the period of negative energy balance (NEB) early post partum. NEFA were then added during in vitro maturation at concentrations measured previously in FF to evaluate their effect on the oocyte's developmental competence. At 16 and 44 days post partum, FF of the dominant follicle and blood were collected from nine high-yielding dairy cows. Samples were analysed for NEFA concentration and composition. NEFA concentrations in FF (0.2-0.6 mmol/l) during NEB remained +/- 40% lower compared with serum (0.4-1.2 mmol/l). The NEFA composition differed significantly between serum and FF with oleic acid (OA), palmitic acid (PA) and stearic acid (SA) being the predominant fatty acids in FF. Based on these results, 5115 oocytes were matured for 24 h in serum-free media with or without (negative control) the addition of 0.200 mmol/l OA, 0.133 mmol/l PA or 0.067 mmol/l SA dissolved in ethanol or ethanol alone (positive control). Matured oocytes were fertilized and cultured for 7 days in SOF medium. Addition of PA or SA during oocyte maturation had negative effects on maturation, fertilization and cleavage rate and blastocyst yield. More (late) apoptotic cumulus cells were observed in cumulus-oocyte complexes matured in the presence of SA or PA. Ethanol or OA had no effect. These in vitro results suggest that NEB may hamper fertility of high-yielding dairy cows through increased NEFA concentrations in FF affecting oocyte quality.

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Non-esterified fatty acids in follicular fluid of dairy cows and their effect on developmental capacity of bovine oocytes in vitro.

The effect of silicone and paraffin oil, which is routinely used to overlay in vitro embryo culture media, on bovine in vitro embryonic development and on their subsequent survival after one-step vitrification was investigated. In experiment 1, silicone oil batch 13 was compared with paraffin oil. Embryonic development to the eight-cell and blastocyst stage was significantly impaired by silicone oil batch 13 in comparison with paraffin oil (p < 0.0001). Normal looking blastocysts produced under both types of oil were vitrified. None of the blastocysts that were produced under silicone oil batch 13 survived the vitrification procedure whereas 59% of the blastocysts survived when they were cultured under paraffin oil both before vitrification and after warming. In experiment 2, another batch no. 7 of the same brand of silicone oil was compared with paraffin oil. No effect of the type of oil on embryonic development until the eight-cell stage could be demonstrated. However, the blastocyst formation rate was significantly lower with silicone oil batch 7 than with paraffin oil. The survival of vitrified blastocysts after warming was significantly improved when silicone batch 13 was replaced by batch 7 (0 and 41%, respectively) although it was still lower when compared with the survival of blastocysts developed under paraffin oil (53%) (p < 0.05). In further attempts to find the cause of the problem, the silicone and paraffin oils were analysed for Zn-contents (experiment 3). Zn-contents were comparable for silicone oil batch 13 (0.87 microM), silicone oil batch 7 (0.62 microM) and paraffin oil (0.73 microM). Media, conditioned by bovine oviduct epithelial cells cultured with a silicone or paraffin oil overlay were analysed by means of thin layer chromatography for differences in qualitative fatty acid composition. It was possible to detect that oil overlay changed the relative abundance of fatty acids in the different lipid classes. In the free fatty acid fraction, it was skewed in favour of palmitic acid and in cholesterol esters and phospholipids in favour of linoleic acid. It appears that due to the changed lipid contents of the medium, embryonic membranes are rendered more susceptible to freezing due to altered membrane composition or by membrane damage caused by lipid peroxidation.

A Christophe

Papers

Non-esterified fatty acids in follicular fluid of dairy cows and their effect on developmental capacity of bovine oocytes in vitro.

Silicone oil used in microdrop culture can affect bovine embryonic development and freezability.