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Mechanisms Linking Metabolic Status and Disease with Reproductive Outcome inthe Dairy Cow

DC Wathes

Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, UK

Contents

Culling for infertility remains the main reason for disposal ofdairy cows, limiting productive lifespan. In extreme cases,ovulation is inhibited, preventing the possibility of conception.More often cows do conceive, but fail to remain pregnantowing to intrinsic problems in the embryo andor to a poor-quality reproductive tract environment. Both aspects have agenetic component and are also influenced by managementpractices affecting nutrition and health. The relative impor-

tance of these factors varies among heifers, first-lactation andolder cows. A common theme, however, is that an internalsignalling system exists which reduces fertility when the cow isin an unsuitable metabolic state to sustain a pregnancy. Thismay be directly related to nutrient shortage caused byinadequate feed intake, or because available nutrients arebeing prioritized towards growth or milk production, awayfrom reproduction. Evidence is presented for the involvementof the somatotrophic axis (GH, IGF1, insulin, IGFBP2) andleptin as key metabolic signalling molecules. Another emergingtheme is the interaction between metabolism and disease thataffects the fertility. Common examples include (i) calf diseasescausing inadequate heifer growth and increased age at firstcalving; (ii) poor peripartum energy status reducing thecapacity of the uterus to involute and mount an effective

immune response, thereby increasing the likelihood of endo-metritis; and (iii) development of mastitis after conception, acontributory factor to both early and late embryo mortality.Finally, recent evidence suggests that times of metabolic stresscause mitochondrial damage that also contributes to areduction in longevity.

Introduction

Holstein dairy cows have an average productive lifespanof approximately three lactations or less (Hare et al.2006). A cow only recoups her rearing costs during hersecond lactation, so profitability improves with in-creased longevity, associated with a greater proportionof total lifetime spent in milk production (Jagannathaet al. 1998). Despite this, approximately 1520% ofdairy cows are culled in their first lactation, mainly dueto poor fertility (Brickell and Wathes 2011). Under-standing the underlying causes of poor fertility requiresa holistic approach, encompassing aspects of reproduc-tive biology, embryology, metabolism, immunology andgenetics. This is being facilitated by recent advances ingenomic technology. Use of gene expression arrays andassociation studies of single-nucleotide polymorphisms(SNPs) with fertility traits has helped to reveal whichbiological pathways are of key importance. This reviewconsiders why failure to conceive and remain pregnant

continues to be the primary reason why so many dairycows worldwide are culled at a relatively young age. Thefocus is on some of the underlying metabolic mecha-

nisms involved and the interactions with disease that caninfluence the fertility.

Causes of Reproductive Failure

Reproductive failure can have multiple causes, summa-rized briefly here. Animals may not be inseminatedbecause of a failure to ovulate (Peter et al. 2009) or todetect oestrus (Saint-Dizier and Chastant-Maillard2012) or because of poor health, low milk yield, poorconformation or an unsuitable temperament. For cowsthat are undergoing oestrous cyclicity, fertilization ratesgenerally exceed 90% (Diskin and Morris 2008), butmany animals do not subsequently remain pregnant.Approximately, 40% of early embryos die in moderateproducing dairy cows, mostly between 8 and 16 dayspost-insemination. This increases to 56% in higheryielding cows, with many embryos already showingabnormal development by day 7 (Diskin and Morris(2008). Several potential causes of early embryo mor-tality have been established. Firstly, oocyte quality maybecome compromised during periods of extreme nega-

tive energy balance (NEB), possibly due to excessaccumulation of non-esterified fatty acids (NEFAs)(Van Hoeck et al. 2011), or toxic effects of elevatedconcentrations of ammonia andor urea associated withhigh protein diets (Leroy et al. 2008). Secondly, avariety of factors can influence the ability of thereproductive tract to support an early pregnancy.Oestrogen and progesterone are key regulators of thetract environment, so anything that affects follicular andluteal development and their ability to secrete sufficienthormone at the appropriate time can also influenceembryo development (Diskin and Morris 2008; Robin-son et al. 2008). There are also metabolic influencesdirectly on tract secretions (Wathes et al. 2008a) andadverse effects of disease status (discussed below).

Additional embryos die slightly later, mainly in thesecond month of gestation. These later losses, whichoccur at a frequency of 720% (Diskin and Morris2008), are more frequent in higher yielding cows andmany may be disease related. Numerous infectiousagents have been associated with embryo mortality andabortion in cattle, including bovine virus diarrhoea virus(BVDV) and Neospora caninum, which are widespreadin many dairy cow populations (Givens and Marley2008). Another potential cause of embryo loss is thedevelopment of clinical mastitis (Hansen et al. 2004).Finally, some bulls and genotypes have been associated

with fertilization failure and others with reducedembryo survival rates (Bulman 1979; Khatib et al.2010). A recent study that transferred in vitro produced

Reprod Dom Anim 47 (Suppl. 4), 304312 (2012); doi: 10.1111/j.1439-0531.2012.02090.x

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blastocysts into either beef heifers or parous dairy cowsconcluded that 30% of early embryo losses wereattributable to the embryo itself (Berg et al. 2010).

Age-related Influences on Fertility

For an individual cow to survive several lactations, shemust remain consistently fertile. Age and lactationnumber both influence fertility, so they need to beaccounted for in models investigating reasons forreproductive failure. Heifer fertility is a key factor indetermining age at first calving (AFC). Animals with adelayed AFC have worse reproductive performance inthe first lactation and reduced longevity (Wathes et al.2008a; Sakaguchi 2011). Respiratory diseases and diar-rhoea are endemic within most populations of dairycalves (Johnson et al. 2011). These contribute to reducedgrowth rates and delayed puberty and prevent animalsfrom reaching rearing targets (Brickell et al. 2009a).

Kuhn et al. (2006) reported a conception rate of 56% inAmerican Holstein heifers: conception rates were max-imal at 1516 months of age, reducing by 13% in olderanimals (26 months).

Dairy heifers should calve for the first time at 2 yearsof age and 8290% mature body weight, so they mustuse nutrients for growth as well as for milk productionduring their first lactation. They therefore differ meta-bolically to multiparous animals. Inadequate growth is arisk factor for maternal dystocia and can have detri-mental influences on fertility in the first lactation(Ettema and Santos 2004). Conversely, late calvingheifers are more likely to become overconditioned, alsoaffecting calving ease. Perinatal mortality, a risk factor

for subsequent poor fertility, is approximately two timesmore likely at first calving (Brickell et al. 2009b).Fertility may be worse in first-lactation cows in com-parison with older animals (e.g. Wu et al. 2012), andthey experience a higher incidence of delayed resump-tion of ovarian cyclicity (Wathes et al. 2007a). Althoughmost older cows have achieved mature body size, theyalso have a greater capacity for milk production. Beta-hydroxybutyrate (BHB) concentrations are higher inearly lactation, associated with the development ofclinical or subclinical ketosis (Duffield et al. 1997;Wathes et al. 2007b). Higher lactation number andprevious lactation milk yield both increase the risk of

retained placenta (Fleischer et al. 2001). Older cows arealso more likely to develop a persistent corpus luteum(Opsomer et al. 2000), and conception to a particularinsemination declines once cows reach 5th parity(Inchaisri et al. 2010).

Tissue Mobilization and Negative EnergyBalance (NEB)

Modern dairy cows experience a period of nutrientshortage (NEB) in early lactation, as body reserves aremobilized to support milk output (Lucy 2001; Watheset al. 2008b). Although the precise relationships are notalways consistent among studies, fertility can be influ-

enced by the body condition score (BCS) before calving,the subsequent rate and extent of mobilization of bodytissue, and the time at which the nadir in body weight is

reached (e.g. Butler 2003; Westwood et al. 2002; Watheset al. 2007a; Sakaguchi 2011). The most important deter-minant of BCS is dry matter intake (Hayirli et al. 2002).These variables are also influenced by dry cow diet(Beever 2006) and the shape of the lactation curve. Somecows quickly reach a high peak milk yield associated

with rapid BCS loss, whereas others achieve a high 305-day milk yield through greater persistency of milkproduction.

Endocrine and Metabolic Factors

Metabolic and endocrine changes associated with nutri-ent shortage and endogenous body tissue mobilizationhave taken on signalling roles that prevent the cowestablishing a pregnancy when conditions are suboptimalvia inhibitory actions at the level of the brain, ovary orreproductive tract. Concentrations of NEFAs and BHBincrease in early lactation, reflecting the extent of adipose

tissue mobilization and fatty acid oxidation, respectively(Bauman and Currie 1980). Glucose concentrations showa short-term decrease at this stage (Bell 1995), while ureaconcentrations may rise or fall, depending on the proteincontent of the diet and the degree of tissue mobilization(Laven et al. 2007). Such metabolite changes can influ-ence reproductive processes directly. In addition, theycontribute to growth-related and post-partum changes inmetabolic hormones that also affect the fertility.

IGF System and Fertility

Increased LH pulsatility during the peripubertal periodpromotes follicular development, leading ultimately to a

sufficient rise in oestradiol production to induce the firstpre-ovulatory LH surge (Day et al. 1987). This usuallyoccurs at approximately 9 months, but only once theheifer possesses an adequate body size andor metabolicstatus to reproduce successfully (Rawlings et al. 2003;Taylor et al. 2004a). Numerous studies (reviewed byVelazquez et al. 2008) have provided evidence thatIGF1 is an important metabolic mediator in the timingof puberty. The circulating IGF1 concentration isclosely related to body weight during pre-pubertalgrowth, and faster-growing, well-fed animals attainpuberty earlier (Macdonald et al. 2007). SystemicIGF1 concentrations in dairy heifers peak shortly before

the onset of puberty, then gradually decline as growthrates decline (Brickell et al. 2009a; Fig. 1a).The start of lactation is also associated with profound

changes in the somatotrophic axis. During metabolicshortages, the growth-promoting actions of GH medi-ated by IGF1 are curtailed and GH instead promotestissue mobilization. This switch is primarily triggered bydown-regulation of the liver-specific variant of the GHreceptor (GHR1A) (Kobayashi et al. 1999; Fenwicket al. 2008a). This in turn is responsible for thepronounced decrease in circulating IGF1 concentra-tions, which begins before calving and typically reachesa nadir in the first week post-partum (Taylor et al.2004b; Fig. 1a).

The actions of IGF1 are influenced by concur-rent changes in IGF-binding proteins (IGFBPs)(Jones and Clemmons 1995). The majority (>90%) of

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hepatic-produced IGF1 normally circulates bound in aternary complex with IGFBP3 and the acid-labilesubunit (ALS). Their production is also regulated bythe hepatic growth hormone receptor (GHR), so bothdecline in early lactation while the production ofIGFBP2 rises (Fenwick et al. 2008a). These changesreduce the half-life of circulating IGF1. The ratio ofIGF1 to IGF2 secreted also changes in the post-partumcow. Hepatic IGF2 production is not regulated by GHor affected by the stage of lactation (Fenwick et al.2008a). Circulating IGF2 concentrations are thus main-tained during periods of nutrient shortage when IGF1concentrations are low. The local production of IGFBPsalso regulates IGF activity. Most tissues express severalIGFBPs that have variable affinities for IGF1 and IGF2(Jones and Clemmons 1995). Both IGF1 and IGF2activate signalling through IGF1R, but the implicationsof a change in ligand for downstream signalling have notbeen addressed. It is thus hard to predict what theoverall outcome will be in terms of the strength of

activation of the IGF1R signalling pathway in aparticular tissue in response to a particular circulatingconcentration of IGF1.

The mechanisms by which IGF1 concentration caninfluence fertility in cattle have been reviewed previously(e.g. Velazquez et al. 2008) and are summarized brieflyhere. IGF1R are present on the pituitary gland, ovariesand reproductive tract. IGF1 can enhance LH secretion(Adam et al. 2000), increase follicular growth and

oestradiol synthesis (Webb et al. 2004), promote uterinehistotroph secretion (Wathes et al. 2008a) and increasethe rate of early embryo development (Block 2007). Indairy heifers, reduced IGF1 concentrations at both 1and 6 months of age were associated with delays in ageat first breeding and so an increase in AFC (Brickellet al. 2009c; Fig. 1c). First-lactation cows that experi-enced a delayed interval to first ovulation post-partumhad lower IGF1 concentrations than their peers at6 months (Taylor et al. 2004a). Cows with a low post-partum nadir in their IGF1 concentration take longer toresume oestrous cycles following calving and are alsoless likely to conceive (Butler 2003; Taylor et al. 2004b;

Patton et al. 2007). This finding is supported by studiesin which bovine somatotrophin injection at the time ofinsemination improved conception rates in repeatbreeder cows (Morales-Roura et al. 2001).

IGFBP2

Although all of the IGFBPs 16 are expressed in theovary andor reproductive tract, our attention hasfocused particularly on IGFBP2, as this binding proteinplays a key role in regulating IGF bioavailability indifferent tissues according to EB status. It is the secondmost abundant IGF-binding protein in the circulation,has high affinity for both IGF1 and IGF2 and is

generally considered to inhibit IGF activity (Jones andClemmons 1995). Circulating concentrations of IGFBP2increase after calving when the expression of the otherIGFBPs is reduced (McGuire et al. 1995). In post-partum cows, hepatic IGFBP2 mRNA expression waspositively correlated with circulating NEFA and BHBand negatively correlated with hepatic glycogen, bloodglucose and IGF1 (Fenwick et al. 2008a). IGFBP2 is akey inhibitor of adipogenesis (Boney et al. 1994), and inhumans, SNPs for IGFBP2 have been linked withdiabetes, obesity and insulin resistance (Grarup et al.2007). We have shown that IGFBP2 mRNA expressionin ovarian granulosa cells and the oviduct decreased

when cows were in severe NEB in early lactation, incontrast to the hepatic up-regulation at this time(Llewellyn et al. 2007; Fenwick et al. 2008b).

We therefore investigated the associations betweenSNPs in the bovine IGFBP2 gene with growth, fertility,milk production and metabolic traits in dairy cows(Clempson et al. 2012). Heifers with the TT and CCgenotypes of BP2_2 were significantly older thanheterozygotes at first conception and subsequentlyproduced higher 305-day milk yields than the hetero-zygotes, even though their lactations were shorter. TheIGFBP2 SNP genotype was also associated with circu-lating glucose, insulin and BHB concentrations aroundcalving. Associations of IGFBP2 SNPs with growth

traits have also been reported in chickens and pigs (Leiet al. 2005; Mote and Rothschild 2006). Differentialregulation of IGFBP2 production by metabolic signals

Bod

yweight(kg)

IGF1(ng/ml)

(a)

Bodyweight(kg)

0 6 12 18 24 30 36

Age (months)(b)

(c)

IGF1(ng/ml)

Fig. 1. (a) Changes in body weight (solid line) and circulating IGF1(dashed line) in dairy heifers (n = 387) with age during their initialgrowth phase and around their first and second calving (arrows). (b)The body weight and (c) the circulating IGF1 concentration of theheifers at 1 and 6 months of age according to their age at first calving(AFC). Animals that were lighter and had lower IGF1 concentrationsat these time points subsequently calved later, **p < 0.01:AFC30 month (n = 36)

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in different tissues may thus control the availability ofIGF1 and IGF2 to activate the IGF1R and so modulatethe growth and reproduction with respect to nutrientavailability.

InsulinInsulin has an important influence on nutrient parti-tioning. Decreasing insulin concentrations together withelevated placental lactogen levels in late gestation triggeradipose mobilization (Bell 1995). In the lactating mul-tiparous cow, insulin concentrations are low post-partum and are negatively correlated with milk yield(Wathes et al. 2007b). Insulin infusion promotes anincrease in hepatic GHR1A and IGF1 production aftercalving (Butler 2003). Relationships between the circu-lating insulin concentration and fertility outcomes havebeen demonstrated in beef cross heifers and lactatingcows fed diets designed to enhance or reduce insulin

secretion (Adamiak et al. 2005; Garnsworthy et al.2009). However, for animals on more normal diets, wehave failed to establish a relationship between insulinlevels and fertility in either dairy heifers (Brickell et al.2009c) or lactating cows (Wathes et al. 2007a). Insulinmeasurements are not as useful as IGF1 in predictingfertility outcomes for several reasons. They are stronglyinfluenced by time in relation to feeding, and both hypo-and hyperinsulinaemia are associated with poor fertilityoutcomes. In addition, the main point of control ininsulin signalling is at the level of the receptor ratherthan the circulating concentration. High lipid concen-trations and acute infections can both cause peripheralinsulin resistance and so reduce glucose uptake into non-

essential tissues during nutrient shortage (Drobny et al.1984; White 2006).

Leptin

Leptin, a product primarily of white adipose tissue,contributes to the regulation of feed intake, energypartitioning and adipose tissue deposition during bothshort- and long-term changes in nutritional state (Ingv-artsen and Boisclair 2001). Circulating leptin concen-trations in cattle are elevated pre-partum and at thisstage are highly correlated with BCS; they decline atcalving and then remain low even when the energy status

has improved (Ingvartsen and Boisclair 2001; Watheset al. 2007b). Peripartum leptin concentrations weresignificantly higher in primiparous than in multiparouscows (Wathes et al. 2007b). Post-partum hypoleptina-emia may promote voluntary feed intake and contrib-utes to peripheral insulin resistance (Ingvartsen andBoisclair 2001).

With respect to possible direct effects on reproduc-tion, leptin receptors are present in the bovine follicleand corpus luteum (Spicer 2001) and endometrium(Thorn et al. 2007). Leptin concentrations in cattleincrease prior to puberty (Thorn et al. 2007) and mayneed to reach an adequate threshold level for theattainment of puberty (Cunningham et al. 1999). Two

studies have shown associations between leptin poly-morphisms with calving difficulty and perinatal calfmortality, suggesting that problems in leptin signalling

pre-partum may compromise placental and foetal devel-opment (Brickell et al. 2010; Giblin et al. 2010). A highleptin concentration before calving was a strong predic-tor of a delayed start to cyclicity and longer intervals toconception, but only in multiparous rather than inprimiparous cows (Wathes et al. 2007a). Other studies

have reported that low leptin concentrations aftercalving may contribute to long intervals to first ovula-tion (Liefers et al. 2005).

In ruminants, severe undernutrition is needed for leptinto influence gonadotrophin secretion, so this effect mayonly be important in extreme circumstances (Zieba et al.2005). Leptin can, however, affect the ovaries directly.Leptin promoted oocyte maturation in vitro, increasingboth the fertilization rate and the proportion of embryosdeveloping to the blastocyst stage (Boelhauve et al.2005).While leptin alone had little effect on ovarian steroido-genesis, high leptin concentrations inhibited insulin orIGF1-stimulated oestradiol production in cultured gran-

ulosa cells (Spicer 2001), but had a synergistic effect withIGF1to promote lutealprogesterone production(Nicklinet al. 2007). Recent work on cancer cell lines has shownsignificant interactions between intracellular actions ofleptin and IGF (Saxena et al. 2008). Variations incirculating concentrations of leptin and IGFs associatedwith BCS and parity could therefore act synergistically toinfluence reproductive tissues via cross-talk between theirrespective signalling pathways.

We recently reported that several leptin gene poly-morphisms were associated with fertility traits in dairycows (Clempson et al. 2011a). In lactating cows, thisincluded effects on days to conception and consequentlycalving interval. Previous studies in cattle have associ-

ated SNPs in leptin and should be its receptor with avariety of milk production traits and with dry matterintake (Liefers et al. 2005). Altered leptin activity couldtherefore have indirect effects on fertility via changes inenergy balance status. Heifer fertility traits were, how-ever, also affected in our study, including the number ofservices needed and AFC. This suggested that someactions on fertility are direct, possibly via the effects onthe ovary and oocyte described above.

Mitochondria and Metabolic Rate

Surprisingly, little attention to date has focused on

mitochondrial activity in the dairy cow. Mitochondriaplay a key role in intracellular energy production andare the main site of intracellular oxygen consumption.Consequently, they also produce reactive oxygen species(ROS) as a by-product of the electron transport chain.In high concentrations, ROS are harmful, causingdamage to both proteins and DNA (Mammucari andRizzuto 2010). Cells initially remove damaged organ-elles by autophagy, including breakdown of the mito-chondria themselves (mitophagy), promoting themaintenance of a functional mitochondrial populationand providing additional nutrients to the cell in times ofshortage (Mammucari and Rizzuto 2010). When moreextreme damage occurs, changes within the mitochon-

dria instead promote apoptosis. The switch to anapoptotic pathway may be encouraged by growth factorwithdrawal and increasing activity of p53, which senses



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intracellular stress signals including DNA damage,hypoxia and nutrient shortage (Mammucari and Rizz-uto 2010).

In our studies on the effects of severe NEB on tissuesof the post-partum cow using gene expression arrays(Fenwick et al. 2008a,b, McCarthy et al. 2010), only a

few genes were consistently up-regulated across alltissues. These included PDK4 and TIEG1 in oviduct,uterus and liver (D. C. Wathes, M. Fenwick and Z.Cheng, unpublished observations). As discussed above,glucose is scarce after calving, but there is ampleavailability of long-chain fatty acids. PD4K is a mito-chondrial enzyme that is up-regulated in response to anincreased lipid supply, inactivating the pyruvate dehy-drogenase complex and helping to conserve glucose bylimiting the conversion of pyruvate to acetyl-CoA(Holness and Sugden 2003). TIEG1 is a transcriptionfactor that can induce apoptosis via the mitochondrialpathway (Jin et al. 2007). TIEG1 is modified by the O-

GlcNAc pathway, which can reversibly alter proteinactivity according to the glucose availability (Alemuet al. 2011).

We also investigated the associations between SNPs intwo autosomal mitochondrial genes with fertility andmilk production traits. TFAM encodes a histone-likeprotein essential for transcription and replication ofmitochondrial DNA (Jiang et al. 2005). Uncouplingproteins (UCPs) transport protons across the innermitochondrial membrane, contributing to the regulationof energy metabolism and the attenuation of ROSproduction (Echtay 2007). In beef cattle, polymorphismsinTFAMand UCP2have been associated with subcuta-neous fat depth, marbling and body weight (Jiang et al.

2005; Sherman et al. 2008). In the UK, Holstein-Friesiancows that were GG homozygotes for TFAM3 were lesslikely to conceive than heterozygotes, had a 24-day longercalving interval and produced less milk (Clempson et al.2011b). They were also more likely to be culled or die,particularly during the second lactation, so fewer GGhomozygotes survived into a third lactation(Fig. 2a). TheAA homozygotes also had slightly worse fertility. Only33% of GG homozygotes for TFAM3 and 30% of AAhomozygotes in the study completed a second lactation,compared to 44% of the heterozygotes (p < 0.05).Infertility was the main reason for culling.

Most cows in our population were homozygous

(GG) for UCP2, with 6% of CG heterozygotes and noCC animals present. The heterozygotes had a reducedage at first conception and a delayed return to cyclicityafter first calving when compared to the homozygotes.However, proportionately more GG than CG animalswere culled before third calving (64 vs 37%, p < 0.05)(Clempson et al. 2011b; Fig. 2b). Single-nucleotidepolymorphisms in both TFAM and UCP2 were alsoassociated with growth traits in the heifers, but therelationship with survival only became evident oncethe animals had started to produce milk. It is aninteresting possibility that modifications to these pro-teins affect the ability of mitochondria to adapt to thechanges in energy supply required at the start of

lactation (Fig. 3). Excessive ROS damage at this timemay then damage a variety of tissues, promotingdisease and decreasing both fertility and longevity.

Metabolism and Disease

The transition from late pregnancy to early lactation isalso associated with a compromised immune status (Caiet al. 1994; Mallard et al. 1998). Mounting an effectiveimmune defence is energetically demanding (Fox et al.

2005), so during infection nutrients are targeted awayfrom other body functions towards the immune system(Spurlock 1997). Both the level of impairment and therate of recovery of immune capabilities post-partum arethus strongly influenced by the extent of NEB aroundcalving (Pyo ra la 2008; Wathes et al. 2009). This makes

TFAM3(a)

(b) UCP2

Fig. 2. KaplanMeier analysis showing the proportion of animalssurviving from birth through to the end of the second lactationaccording to (a) the TFAM3 single-nucleotide polymorphism (SNP)and (b) theUCP2SNP (from Clempson et al. 2011b, with permission)

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high-yielding cows in NEB susceptible to infection fromthe multiplicity of pathogenic organisms commonlypresent in the farm environment. Within 2 weeks aftercalving, 40% of cows develop metritis, whereas endo-metritis andor mastitis are present in approximately 15and 2050% of all dairy cows, respectively (Zhao and

Lacasse 2008; Sheldon et al. 2009).Endometritis and mastitis are therefore extremely

common and are also known to decrease fertility. Notonly do ongoing infections disrupt the pre-ovulatory LHsurge and inhibit normal follicular maturation (Sheldonet al. 2009), but an impaired uterine environment isalmost certainly a major factor in repeat breeder cowsthat have a higher incidence of early embryonic death(Hill and Gilbert 2008). Mastitis is also associated with areduction in pregnancy rate (Hansen et al. 2004; Schricket al. 2011).

There is a complex inter-relationship between acuteinfection, a predisposition to chronic inflammation and areduced capacity for tissue repair. This is of particularimportance in the uterus, which must undergo extensiveremodelling after calving before it is ready to establishanother pregnancy. Using arrays to compare geneexpression in endometrium of post-partum cows in severeor moderate NEB, we found differential expression of 240genes, nearly half of which were associated with immuneor inflammatory pathways (Wathes et al. 2009). Therewere also changes in the expression of genes associatedwith the IGF and insulin signalling pathways includingIGFBP6, IGF1 and IGFALS which were all reduced.However, AHSG and PDK4, two genes implicated ininsulin resistance, were more highly expressed in theendometrium of SNEB cows. As IGF1 has a positive

effect on tissue repair mechanisms following injury(Mourkioti and Rosenthal 2005), our data supportevidence that uterine involution and elimination of

bacteria are delayed when cows are in NEB (Lewis1997). This predisposes them to develop subclinicalendometritis; affected animals have a markedly reducedrate of conception, and the proportion which fail toconceive at all also rises significantly (Gilbert et al. 2005).

Conclusions

Infertility problems in dairy cattle are multifactorial andare associated with both genetics and management.Individual cow factors relating to the age and health ofthe animals, the amount of feed consumed and how it isutilized internally (nutrient partitioning) influence theirability to conceive and remain pregnant. While each cowis bred with a genetic potential to achieve a certain levelof milk production, this can only be realized if she isprovided with a lifetime environment which enables herto fulfil her potential; this requires her to remain fertile.Improvements in fertility can be achieved in the short-term by identifying the main causes of infertility in aparticular herd and adoption of optimized managementstrategies involving nutrition, reproductive managementand animal health. A longer-term sustained improve-ment in fertility must also encompass appropriategenetic selection to identify females with high fertilitytraits using molecular genetic technologies.

Acknowledgements

I am very grateful to my many excellent colleagues who havecontributed to the research performed at the Royal Veterinary Collegeand to DairyCo, Defra, Merial Animal Health Ltd., Volac Interna-tional Ltd., BBSRC and the Wellcome Trust who helped to fund it.

Conflicts of interest

The author does not have any conflicts of interest to declare.

mtDNA

Mitochondrial

proteins

Proteins imported from cytosol

Electron transport chain

NADH NAD+ O2 H2O

heat

ROS

mitophagy

apoptosis

Growth

factor

withdrawal

eg reduced

IGF1

Cellular stress

(hypoxia, DNA

damage, nutrient

shortage)

p53

NEB

switches

energy

supply

from

glucose

to NEFA

PDK4

TIEG1

mtDNAreplicaon

TFAM3

UCP2

Fig. 3. Summary diagram of the impact of negative energy balance (NEB) on the mitochondrial population. As the supply of glucose is reduced,PDK4 is up-regulated to promote the use of fatty acids for energy production. As energy production increases, more reactive oxygen species(ROS) are produced as a by-product of the electron transport chain. This damages both mitochondrial proteins and DNA. Damagedmitochondria are removed by mitophagy or apoptosis. The balance towards apoptosis is tipped by changes in whole-body metabolism reducingIGF1 concentrations, by increasing cellular stress acting through the p53 pathway and by up-regulation of the transcription factor TIEG1influencing gene expression. The ability of the remaining mitochondria to replicate and replace those which have been destroyed is influenced bythe genotype for TFAM3, while the UCP2 genotype may be important for the attenuation of ROS production, so limiting the initial damage



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Authors address (for correspondences): DCWathes, Royal Veterinary College, Hawks-head Lane, North Mymms, Hatfield, HertsAL9 7TA, UK. E-mail: [email protected]

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