Declining fertility, insulin resistance and fatty acid metabolism in ...

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Acta Scientiae Veterinariae. 38(Supl 2): s545-s557, 2010.

ISSN 1678-0345 (Print)ISSN 1679-9216 (Online)

Declining fertility, insulin resistance and fatty acid metabolismin dairy cows:

Developmental consequences for the oocyte and pre-implantationembryo

Kevin D Sinclair1

ABSTRACT

Background: The fertility of contemporary high-yielding dairy cows is in decline and herd health and lifespan aredecreasing. Over-conditioned peri-parturient cows are more insulin resistant than lean cows and this leads to anincrease in body fat mobilisation, in the form of free fatty acids (FFA). This, in turn, can increase the risk of a numberof disorders including fatty liver syndrome, ketosis, milk fever and infertility. In humans, the accumulation of fat innon-adipose tissues and excess accumulation of fat in visceral adipose depots also contribute to peripheral insulinresistance. The delivery of FFA to the liver from visceral adipose tissue is now believed to be the primary contributorto the pathological symptoms of hepatic lipidosis, hyperinsulinaemia and glucose intolerance. The current articledevelops the hypothesis that the modern high-yielding dairy cow has inadvertently been selected to become increasinglyinsulin resistant, in part, as a consequence of a regional re-distribution in body fat towards intra-abdominal depots. Inturn, this contributes to greater lipolysis and mobilisation of FFA during early lactation with implications for cowmetabolism and fertility. These ideas are developed in the context of recent data on the effects of insulin and fattyacids on oocyte and early embryo development, with reference to mice and humans where mechanistic insights fromruminants are lacking.

Review: Relative to non-ruminants, cattle and sheep are insulin resistant, but the mechanisms of insulin action inruminants are believed to be similar to those of other species. Key insulin signalling pathways have been identified inboth physiological and pathological conditions in humans and mice, but these mechanisms are poorly understood inruminants. FFA are key inflammatory signals which, together with specific adipokines, can induce insulin resistance.Again, the mechanisms of action for these factors are poorly understood in ruminants. However, obesity is associatedwith hyperinsulinaemia and impaired egg quality in all species studied. Insulin sensitising agents in obese mice havebeen found to improve the post-fertilisation developmental potential of oocytes. In cattle, the detrimental effects ofhyperinsulinaemia on oocyte quality are cumulative over time, suggesting that exposure to high levels of insulinduring pre-antral follicular development is particularly harmful. Insulin resistance can also manifest in the pre-implantationembryo. In the mouse this leads to apoptotic cell death and lower pregnancy rates. The mechanisms of fatty acid-inducedinsulin resistance in the oocyte and embryo are currently not known. However, the follicle-enclosed oocyte preferentiallyaccumulates saturated fatty acids, and high levels of palmitic (c16:0) and stearic (c18:0) acid impair maturation and post-fertilisation development. In contrast, specific polyunsaturated fatty acids (PUFA) (e.g. α-linolenic acid (c18:3n-3)) canimprove oocyte maturation and embryo development in vitro, but the effects of n-6 PUFA are generally inhibitory. However,it has been difficult to reproduce these findings in lactating cows. This is possibly due to selective uptake mechanisms inthe ovary and high levels of relatively saturated FFA in peripheral circulation at this time.

Conclusion: Insulin resistance in modern high-yielding dairy cows contributes to reduced fertility and there is evidencethat this effect presents as reduced oocyte quality and impaired embryo development. Detailed mechanistic studiesin cattle are lacking, but the level and nature of FFA (particularly PUFA) may be key in determining the extent ofinsulin resistance and oocyte/early embryo development.

Keywords: insulin resistance, oocyte, embryo, free fatty acids, polyunsaturated fatty acids.

1 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Leicestershire, UK. CORRESPONDENCE: K.D. Sinclair[[email protected] TEL: + 44 (0)115 951 6053].

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Sinclair KD. 2010. Declining fertility, insulin resistance and fatty acid metabolism in dairy cows: Developmentalconsequences for the oocyte and pre-implantation embryo. Acta Scientiae Veterinariae. 38 (Supl 2): s545-s557

I. INTRODUCTION

II. BODY FAT DISTRIBUTION, INSULIN RESISTANCE AND METABOLIC HEALTH

III. MOLECULAR BASIS OF INSULIN RESISTANCE

IV. HYPERINSULINAEMIA, INSULIN RESISTANCE AND EGG QUALITY

V. HYPERINSULINAEMIA, INSULIN RESISTANCE AND EMBRYO DEVELOPMENT

VI. FATTY ACIDS AND OOCYTE MATURATION

VII. FATTY ACIDS EMBRYO DEVELOPMENT

VIII. FEEDING FATTY ACIDS TO DAIRY COWS

IX. CONCLUSIONS

I. INTRODUCTION

The current article is set against a background of declining fertility [64], reduced herd longevity [17] and impairedliver metabolism [50] associated with increased metabolic stress and health related complications in contemporary high-yielding dairy cows (in particular Holstein cows). In contrast, pure-bred and cross-bred Bos taurus beef cows are inherentlyfertile, relatively healthy and long-lived with fewer problems than those listed for dairy cows [69]. Over the past 30 years inthe UK, a combination of breed substitution (Holstein for Friesian) and within-breed selection within the dairy herd hasdoubled average milk yield per cow, but average survival has declined from 4 to <3 lactations [61], due largely to infertilityas 50% of dairy cows are culled for failure to rebreed [16]. Poor fertility also underlies their 20% contribution to UKatmospheric methane emissions, a consequence of having to maintain a relatively large number of non-productivereplacement heifers [26]. The rapid mobilisation of body fat reserves during early lactation is associated with fertility andhealth problems in dairy cows [8,27,61]. Over-conditioned peri-parturient cows are more insulin resistant than lean cows[36], leading to an increase in serum free fatty acids (FFA). This can further increase insulin resistance [56] and increase therisk of fatty liver syndrome, ketosis, dystocia, retained placenta, metritis, milk fever, mastitis and lameness [31,32,44,50].

Accumulation of fat in non-adipose tissues (in particular liver and muscle) is linked to fat level and is alsohighly associated with peripheral insulin resistance [13]. However, in humans, it is also known that excess accumulationof fat in visceral adipose depots leads to Metabolic Syndrome, a complex combination of medical disorders thatincrease the risk of developing hypertension, cardiovascular disease and type II diabetes [24]. Indeed, it is the deliveryof FFA to the liver from visceral adipose tissue via the hepatic portal vein that is now thought to be the primarycontributor to the pathological symptoms of hepatic lipidosis, hyperinsulinaemia and glucose intolerance [82]. Lipolysisin the rat and human differs between adipose depots, with increased expression of lipolytic genes and lipolytic activityin visceral than subcutaneous fatty tissues [6,49,81].

These observations are significant because there is good evidence to indicate that, relative to contemporarybreeds of beef cow, the modern high-yielding Holstein dairy cow has a disproportionately high amount of intra-abdominal fat.Some 30 years ago it was shown that Friesian dairy cows contained a greater proportion of intra-abdominal and a lowerproportion of subcutaneous, fat than the range of beef and beef x cross dairy cows assessed [86], and there is indirectevidence from a variety of sources to indicate that the proportion of intra-abdominal fat, leading to greater insulin resistancein modern Holstein cows, may be even more extreme than for the British Friesians reported in 1984. For example, Nikkhahet al [52] placed a group of non-pregnant, non-lactating Holstein cows on a low or moderate plane of nutrition for 2 months.At the end of this period body condition score (BCS) was similar between the two groups of animals but omental, mesentericand peri-renal fat mass was 70% greater in the better fed group. Based on a single glucose tolerance test, insulin resistanceappeared to be greater in North-American than the more traditional New Zealand Holstein-Friesian dairy cows in the studyof Chagas et al. [12]. Furthermore, a retrospective analysis of plasma insulin concentrations conducted for Holstein orSimmental x Holstein heifers at the author’s laboratory indicated that the ‘leaner’ (BCS = 2 units; 5 point scale, 1 = lean, 5= obese) Holstein heifers were more insulin resistant than their ‘fatter’ Simmental x Holstein contemporaries (Figure 1).Indeed, with mean plasma insulin concentrations of 40 µIU/ml, these Holstein heifers were hyperinsulinaemic and equivalentto Simmnetal x Holstein heifers of 4 units BCS on a high plane of nutrition equivalent to twice their maintenance requirements[1]. Plasma insulin concentrations were also greater in lactating Holstein cows compared to Beef x Holstein cows despitethe fact that peak milk yields for these latter cows were only approximately 0.25 that of Holstein cows (Figure 2).

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Figure 1. Plasma insulin concentrations for Holstein (BCS = 2.0 units) (A) and Simmental x Holstein (BCS = 3.0 units) (B) heifers on similardiets at similar levels of feeding. Data derived from [1,2,3,62,72,75] using the same insulin radioimmunoassay.

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Figure 2. Plasma insulin concentrations for lactating Holstein cows (A) and Beef x Holstein (predominatly Simmental crosses) cows (B).Data derived from [28,29,30,73,76] using the same radioimmunoassay.

The current article develops the hypothesis that, as a consequence of selection for increased milk yields,the Holstein cow has become increasingly insulin resistant. This may, in part, have arisen as a consequence of aregional re-distribution in body fat towards intra-abdominal depots which, in humans and rodents, are known to bemore insulin resistant than subcutaneous depots. This, in turn, would contribute to greater lipolysis and mobilisationof free fatty acids during early lactation which can affect a number of metabolic and reproductive processes in thecow including direct effects on the oocyte and pre-implantation embryo. The article explores these ideas whilstreviewing recent data on the effects of insulin and fatty acids on oocyte and early embryo development. Reference ismade to studies with mice and other species where mechanistic insights from ruminant studies are lacking.

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II. BODY FAT DISTRIBUTION, INSULIN RESISTANCE AND METABOLIC HEALTH

Euglycaemic-hyperinsulinaemic clamp studies in humans, rats and growing or adult ruminants indicatelower insulin responsiveness of peripheral tissues in ruminants. That is, relative to non-ruminants, cattle and sheepare insulin resistant, with the effect being largely mediated through differences in the post-receptor insulin (INS)cascade, e.g. reduced phosphorylation of insulin receptor substrate 1 (IRS1), leading to reduced activity ofphosphoinositide 3-kinase (PI3K) [66]. Nevertheless, the mechanisms of insulin action in ruminants are similar tothose of other species [71]. Of central importance to the current thesis is that insulin resistance and associatedmetabolic complications are as much a consequence of the regional distribution of body fat as overall body fatness.Recent data comparing the effects of short-term diet restriction in obese-old vs. obese-young rats demonstrated thatreversal of insulin resistance was primarily associated with mesenteric fat reduction and hepatic triglyceride loss [10].Interestingly, the old animals in that study had a refractory response in terms of improved peripheral insulin sensitivityto visceral fat loss, which was associated with a delayed clearance of triglyceride from the liver and a delayedrecovery in hepatic insulin sensitivity; somewhat reminiscent of complications associated with fatty liver in agedmulti-parous compared to young primi-parous cows [5]. It is primarily through effects on liver metabolism and hepaticlipidosis [32,34] that differences in regional distribution of body fat and insulin resistance impinge on cow production,health and fertility.

III. MOLECULAR BASIS OF INSULIN RESISTANCE

Much of our current understanding of the molecular mechanisms of insulin resistance stem from work withhuman cells and from rodents. In humans, adipocyte size is related to the proportion of visceral fat, and is increasedin insulin resistant subjects [78]. Whilst activation of INSR, IRS-1 and protein kinase B (AKT) are unaltered byadipocyte size, insulin sensitive facilitative glucose transporter (SLC2A4) translocation to the plasma membrane isreduced in large adipocytes [22], indicating that insulin mediated glucose uptake by large fat cells, associated withincreased abdominal obesity, is reduced. At a more fundamental level, phosphorylation of IRS proteins (in particularat serine (Ser)307 residues has emerged as probably the key step in insulin signalling in both physiological andpathological conditions such as type II diabetes [7,15], being a target for metabolic and inflammatory stresses thatpromote insulin resistance [77].

Free fatty acids are generally considered to be the key inflammatory stimulus leading to serine phosphorylationof IRS1 through the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), as well as c-jun N-terminal kinase (JNK) and S6K1 kinase [15,25]. The innate immune system through Toll-like receptor 4 (TLR4)activation acts as a key nutritional “sensor” whereby FFA may contribute to the pathogenesis of lipid-induced insulinresistance [70]. Adipose tissue also secretes proteins which can influence insulin sensitivity. Peroxisome proliferator-activated receptor-g (PPARG) is highly expressed in adipose tissue and acts as a transcription factor regulating theexpression and secretion of several such adipokines (e.g. adiponectin, interleukin (IL)-6, tumour necrosis factor(TNF), moncoyte chemotactic protein-1 (MCP-1)) [58]. Transcripts for PPARG and its cofactor PPARG coactivator-1a(PPARGC1A) are expressed in both human subcutaneous and omental adipose tissues, but expression of PPARGC1AmRNA is significantly lower in omental than subcutaneous depots, and is generally reduced in adipose tissue ofinsulin resistant subjects [65]. Indeed, a defining property of visceral adipose tissue in insulin resistant women is itspro-inflammatory, pro-thrombotic propensity. IL-6 and -8, MCP-1 and plasminogen activator inhibitor 1, are all greaterin visceral than subcutaneous adipose tissue in insulin resistant subjects [39]. TNF is known to induce insulinresistance in obese mice, in part, by reducing insulin-stimulated autophosphorylation of INSR [80]. The mechanismin adipocytes involves serine phosphorylation of IRS-2 mediated by TNF induced activation of mitogen activatedprotein kinases (MAPKs) whereas, in myotubes, the site of action for TNF is Ser307 phosphorylation of IRS1-mediated by p38 MAPK and inhibitor kB kinase [51]. That serum TNF correlates with insulin resistance and associatedfatty liver in the dairy cow [53], however, is about the extent of our understanding for the species of interest in thecurrent study, emphasising the urgent need to develop an improved understanding of the mechanisms of insulinaction and tissue resistance in this species.

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IV. HYPERINSULINAEMIA, INSULIN RESISTANCE AND EGG QUALITY

A contributing factor to the lower pregnancy and increased miscarriage rates encountered by overweight(BMI ³ 25 kg m-2) women undergoing fertility treatment by IVF [41] may be impaired egg quality associated with insulinresistance. Unfortunately, much of the human data in this area is derived from patients with PCOS [37]. However,recently, the ability of insulin sensitising agents 5-aminoimidazole 4-carboxamide-riboside (AICAR), sodium salicylateand rosiglitazone to enhance the post-fertilisation developmental potential of oocytes was determined in obeseC57BL/6 mice offered a high-fat diet [47]. Rosiglitazone, a potent agonist for the nuclear receptor PPARG, was mosteffective in lowering blood insulin and triglyceride concentrations, and restoring post-fertilisation development of invivo derived zygotes cultured in vitro. Within the mouse ovary PPARG is most highly expressed in granulosa cells[48], where it can interact with target genes such as Cd36 and the HDL receptor, scavenger receptor class B member1 (Scarb1), involved in lipid uptake and metabolism [47].

In cattle, both insulin and insulin-like growth factor (IGF)-I interact synergistically with gonadotrophins topromote follicular growth and steriodogenesis within the follicle. A high energy diet in the study of Armstrong et al [3]significantly increased plasma insulin and IGF-I concentrations, relative to the low energy diet, and increased growthrate and maximum size of the pre-ovulatory follicle. However, increased global nutrient supply reduced oocyte quality,defined as the proportion of oocytes which, following IVF, developed to the blastocyst stage in vitro. In addition toincreases in peripheral insulin and IGF-I, the high energy diet reduced steady-state concentrations of mRNA encodingIGFBP2, IGFBP4, INSR and type 1 IGF receptor in granulosa and thecal cells, changes expected to increase thebioavailability of intra-follicular IGF. The study concluded that diets formulated to optimise follicle development maycompromise oocyte quality. We have since shown that antral follicle development and egg quality are both impairedin obese and hyperinsulinaemic (> 37 mIU/ml plasma insulin) beef x dairy cattle (Figure 3) [1]. In that study, oocyteswere retrieved from donors using ultrasound guided follicular aspiration, and matured, fertilised and cultured in vitro.Further detailed analysis revealed that the negative relationship between insulin and egg quality (defined as theproportion of inseminated oocytes that developed to the blastocyst stage) increased over time; i.e. as the animalsbecame fatter. This effect could have been due to the duration of exposure of oocytes to elevated levels of insulin, butthe data also suggest that oocytes exposed to high levels of insulin during the pre-antral stages of follicular developmentmay be most sensitive to the negative effects of this hormone. Studies in France, also working with overfed heifers,broadly concur with our data, where the benefits of a short-term period of dietary restriction on the post-fertilisationdevelopmental of oocytes have been demonstrated [23].

V. HYPERINSULINAEMIA, INSULIN RESISTANCE AND EMBRYO DEVELOPMENT

Insulin resistance can also manifest in the pre-implantation embryo. Chronic exposure of mouse embryosto either insulin or IGF-I leads to decreased IGF-I receptor expression, impaired insulin-stimulated glucose uptakeand apoptosis [14,54]. Insulin-resistant embryos, as a consequence, undergo apoptotic cell death, manifesting inlower implantation rates when transferred to pseudo-pregnant females [55]. Such embryos exhibit diminished expressionof the insulin-stimulated glucose transporter SLC2A8 which, in the mouse embryo, is expressed from the 8-cell stageonwards [9]. Inhibition of the P13K/Akt pathway in the mouse pre-implantation embryo also leads to reduced insulin-stimulated glucose uptake and apoptosis, which manifest as decreased fetal implantations and increased fetalresorptions [59,60]. AMP-activated protein kinase (AMPK) is a fuel-sensing heterotrimetric kinase which serves as aregulator in glucose and fatty acid metabolism. Cross-talk between AMPK and the PI3K/Akt/mTOR insulin signallingpathway is implicated in insulin-resistant mouse embryos [40]. Knocking down igf1r expression by siRNA decreasedAMPK activity in mouse trophoblast stem cells and reduced 2-deoxyglucose uptake in that study. These effects werereversed, and SLC2A8 translocation to the plasma membrane increased, by AMPK activators AICAR and phenformin.Exposure of mouse pre-implantation embryos to supra-physiological concentrations of insulin during culture has alsobeen found to increase the development of surviving pups following transfer to pseudo-pregnant recipients [68]. Thiswas associated with increased expression of two imprinted genes (i.e. H19 and Igf2) and a loss of methylation at theH19-Igf2 imprint control region.

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Figure 3. Data are for Simmental x Holstein heifers of moderately high BCS (3.75 rising to 4.0 units) fed at twice maintenance (1000 kJME.kg-1.Wt-1.d-1) [1]. Plasma insulin concentrations are either in the ‘Normal’ (N) range (£ 37 µIU.ml.-1) or > 3 SD above the mean of ‘normal’plasma insulin concentrations (hyperinsulinaemic (Hyper or H)) (> 37 µIU.ml.-1). Mean plasma insulin concentrations (A), number of small,medium and large follicles (B), oocytes matured (C) and blastocyst yields.

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VI. FATTY ACIDS AND OOCYTE MATURATION

The mechanisms of fatty acid-induced insulin resistance were alluded to earlier. In the cow plasma FFAconcentrations increase during negative energy balance associated with early lactation, and this source of relativelysaturated fatty acids [38] is readily taken up by cells within the ovary. Indeed, in contrast to surrounding somatic cellsof the ovary, oocytes appear to preferentially accumulate saturated as opposed to unsaturated FA [2,85]. This hintsto selective uptake mechanisms and/or de novo synthesis within the ovarian follicle and oocyte that favours saturatedFA. Lipase specific activity in bovine oocytes is also greater than that of surrounding cumulus cells and increasesduring oocyte maturation [11], hinting to an important role of acquired and endogenous lipid reserves in oocyte energymetabolism during maturation. Such compositional differences between cells within the ovary, however, may merelyreflect differences in triglyceride, phospholipid and cholesterol ester levels associated with the relative prevalence ofcytoplasmic lipid droplets in oocytes and cell membranes in surrounding somatic cells. The uptake and metabolismof FA by the follicle-enclosed oocyte is an area that requires further study. Nevertheless, the FFA composition ofserum and follicular fluid are broadly similar and FFA are rich in palmitic (c16:0), stearic (c18:0) and oleic (c18:1n-9)acids [38]. In that study, the addition of physiological concentrations of either palmitic or stearic acid to bovine oocytematuration media reduced the proportion of inseminated oocytes that cleaved following insemination, and the proportionof zygotes that subsequently developed to the blastocyst stage. The inclusion oleic acid was without effect. Incontrast, the inclusion of physiological concentrations of the polyunsaturated fatty acid (PUFA) alpha linolenic acid(c18:3n-3) [42] but not linoleic acid (c18:2n-2) [43] improved bovine oocyte maturation and post-fertilisation developmentto the blastocyst stage in vitro. The mechanisms associated with the positive response of á-linolenic acid were shownto involve enhanced prostaglandin E

2 production by cumulus-oocyte complexes (COC), increased intracellular

concentrations of cAMP within the COC, and phosphorylation of mitogen-activated protein kinases (MAPK) in oocytes.In contrast, the mechanism associated with the negative effects of linoleic acid involved reduced intracellularconcentrations of cAMP within the COC and reduced phosphorylation MAPK1 and 3, and AKT in the oocyte. Collectively,these mechanisms are known to modulate nuclear maturation and the proportion of metaphase II oocytes for fertilisation.

The fatty acid (FA) profile of both granulosa cells and oocytes can be influenced by maternal diet. Recently,we found that the FA profile of both granulosa cells and oocytes from non-lactating ewes offered either n-3 or n-6 PUFAenriched diets reflected both plasma and dietary levels of these FA, although once again the proportion of saturated FA(mostly stearic acid (C18:0)) was greater in oocytes than granulosa cells [85]. Ewes fed diets supplemented withcalcium soaps of fish oil fatty acids (rich in eicosapentaenoic acid (EPA; c20:5n-3) and docosahexaenoic acid (DHA;c22:6n-3)) produced higher “quality” oocytes that were less sensitive to chilling damage [87]. This was associated withan increase proportion of both EPA and DHA in the phospholipid fraction of cumulus cells but not oocytes; failure todetect PUFA in oocytes was probably due to detection limits of their instruments. In contrast, mice fed a diet enrichedin long chain n-3 PUFA produced inferior quality oocytes associated with impaired mitochomdrial function and enhancedproduction of reactive oxygen species, culminating in reduced post-fertilisation development to the blastocyst stage[83]. At present there are insufficient data to reconcile these apparent discrepancies of n-3PUFA in oocytes; the answermay lie in the apparently antagonistic effects of specific n-3 and n-6 PUFA and requires further investigation.

VII. FATTY ACIDS EMBRYO DEVELOPMENT

The b-oxidation of fatty acids is thought to generate much of the water and at least some of the energynecessary for blastocoel formation [84]. Fergusson and Leese [18] reported a 42% reduction in triglyceride content bythe 2-cell stage in bovine embryos, but there was no subsequent net change in triglyceride content to the hatchedblastocyst stage. However, by maturing oocytes and culturing zygotes in the presence of various concentrations ofthe mitochondrial carnitine palmitoyltransferase A inhibitor, methyl palmoxirate (MP), these authors were able todemonstrate both independent and additive effects of impaired fatty acid oxidation on early post-fertilisation development[19]. By blocking the entry of FA from triglycerides into mitochondria, MP induced a dose dependent reduction in theproportion of oocytes that cleaved following fertilisation and the proportion of zygotes that developed to the blastocyststage. A retrospective analysis of our own data in sheep [84] reveals a similar decrease in total fatty acids duringoocyte maturation, with little subsequent change up to the blastocyst stage (Table 1). In sheep oocytes around 44%of the triglyceride fraction is composed of MUFA of which oleic acid (c18:1n-9) comprises around 75% [46]. Thereduction in percentage MUFA in MII oocytes depicted in Table 1, therefore, probably reflects the extensive oxidationof triglycerides that take place during oocyte maturation.

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Table 1. Fatty acid content and composition (percentage total fatty acids) of immature (germinal vesicle intact)and mature (metaphase II) sheep oocytes and Day 6 embryos (blastocysts) cultured in vitro in the presence offatty acid free bovine serum albumin. Data calculated from [85].

Oocyte

Germinal vesicle Metapahse II Blastocyst

A. Total fatty acids (ng) 89 65 62

B. Percentage total fatty acids

Saturated 56 76 30

Monounsaturated 32 10 25

Polyunsaturated 12 14 44

During pre-elongation development up to the blastocyst stage there is not net increase in embryo mass,although cell number increases to around 120 [74]. This increase in cell number represents a 60-fold increase in cellsurface area that necessitates a significant amount of plasma membrane synthesis. Therefore, the increase inpercentage PUFA at the expense of saturated FA in Day 6 sheep blastocysts (Table 1) probably reflects the increasein cellular phospholipids at the expense of more saturated cholesterol esters and free fatty acids, although suchcompositional changes have yet to be properly established. In contrast to the oocyte, little is known about thecomposition of phospholipids in pre-elongation mammalian embryos, how it might change during development andhow it is affected by maternal diet. It is generally recognised, however, that phospholipids are the major structuralcomponents of all membranes with negligible pools of free phospholipids in the cell [63]. Synthesis of phospholipidduring early pre-implantation development has been described in the mouse embryo using [methyl-3H]-choline as aspecific precursor [57]. [Methyl-3H]-choline incorporation into lipid increased 9-13-fold up to the morula stage, andincreased further up to the blastocyst stage, although the relative extent was difficult to fully quantify in that study.Working with discarded human embryos, Haggarty et al [35] reported that 6-cell to blastocyst stage embryos hadproportionally more PUFA (mostly linoleic acid (C18:2n-6)) than < 6-cell stage embryos. Furthermore, in contrast toisotopically labelled palmitic acid (C16:0), the uptake of labelled linoleic acid increased dramatically beyond the 8-cellstage.

We recently determined the effects of culturing sheep zygotes in SOF media supplemented with high-density lipoproteins (HDL) fractionated from sera of ewes offered n-3 or n-6 PUFA enriched diets [85]. The principalsources of n-3 PUFAs in the experimental diets were salmon oil and linseed, where as the principal source of n-6PUFA was sunflower oil. HDL was added to culture media at physiological levels. Control embryos were cultured inSOF with FA-free BSA. In spite of the fact that sheep embryos expressed transcripts for the HDL receptor SCARB1,fatty acid analysis of Day 6 blastocysts revealed that there was no net uptake of FA from HDL by embryos. The FAcomposition of Day 6 blastocysts was unaltered by culture treatment. Nevertheless, n-6 PUFA HDL significantlyreduced embryo development and transcript expression for genes encoding the LDL receptor and stearoyl-CoAdesaturase relative to either FA-free BSA or n-3PUFA HDL treatments. The mechanisms underlying these effectsremain to be determined. However, the most abundant FA in Day 6 sheep blastocysts, at 34g/100g total fatty acids,was linoleic acid (C18:2n-6). The mass of this FA in blastocysts was greater than that measured in oocytes andindicates that the sheep embryo, like the human embryo [35], has an absolute requirement for linoleic acid which itacquires, at least in our system, from albumin as opposed to HDL.

VIII. FEEDING FATTY ACIDS TO DAIRY COWS

In a study designed to investigate short-term effects of level of rumen inert fatty acids on developmentalcompetence of oocytes in lactating dairy cows, Fouladi-Nashta et al. [20] compared diets supplying 200 or 800 gCaPFA/d offered for several weeks prior to oocyte recovery and in vitro maturation fertilisation and culture to theblastocyst stage. The high-fat diet significantly improved blastocyst production (38 versus 29% of cleaved embryos;P = 0.017) and the total number of cells within each blastocyst at day 8 post-fertilization (151 versus 133; P = 0.043).

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As discussed earlier, in the lactating dairy cow mechanisms within the ovary may be effective in partiallynullifying dietary treatment induced differences in plasma saturated and unsaturated FA composition. Fouladi-Nashtaet al. [21] offered lactating Holstein cows from Day 42 of lactation one of three isocaloric diets that contained (i) arumen inert source of FA rich in plamitic (c16:0) and oleic (c18:1n-9) acids, (ii) full-fat soya (rich in linoleic acid(c18:2n-6) or (iii) linseed, full-fat extruded (rich in alpha linolenic acid (c18:3n-3)). Whilst differences in the FA compositionof both plasma and milk reflected that of the three diets, neither the FA composition of granulosa cells nor the post-fertilisation developmental competence of oocytes was altered by diet. The studies of Fouladi-Nashta et al. [20,21],therefore, suggest that level of dietary fat is more important than type of dietary fat in determining oocyte developmentalcompetence in high-yielding dairy cows. This conclusion is supported by the study of Bilby et al. [4], which found nosignificant difference in blastocyst yield when comparing sunflower oil (high in oleic and n-6 fatty acids), calcium saltsof trans fatty acids, calcium salts of vegetable oil (high in n-6 fatty acids) and linseed oil (high in n-3 fatty acids).Similarly, Thangavelu et al. [79] found no difference in the number of transferable embryos recovered from cows fedsupplements of saturated fatty acids, whole linseed or sunflower seed. As alluded to earlier, the physiological statusof these animals may have contributed to this outcome and, indeed, towards the general inconsistency in responsesto dietary sources of PUFA between studies [45,67]. Peripheral blood in lactating dairy cows will contain a mixture ofFA of dietary origin and from body-tissue catabolism; the latter will contain a high proportion of saturated FA.

IX. CONCLUSIONS

The current article indicates that, in contrast to the human and rodent, a detailed understanding of themechanisms of insulin action, insulin resistance and hyperinsulineamia in cattle is lacking. However, hyperinsulinaemiain cattle is associated with impaired oocyte quality and embryo development. Mechanistic insights into this phenomenonexist in the mouse, where the role of FFA has been identified. FFA in cattle contain a high proportion of saturated FAand these have been shown to impair oocyte maturation and embryo development. In contrast, specific n-3 PUFAhave been shown to improve oocyte maturation and post-fertilisation development in vitro, but it has been difficult toconsistently reproduce these effects in vivo with lactating dairy cows. Continued selection for increased milk yieldhas resulted in modern dairy cows that are increasingly insulin resistant. Consequently, there is an urgent need toprovide a greater mechanistic insight into this phenomenon, so that preventative and remedial strategies can bedeveloped to alleviate this problem.

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