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

x ISSN 0936-6768

Mechanisms Linking Metabolic Status and Disease with Reproductive Outcome in the Dairy Cow
DC Wathes
Royal Veterinary College, Hawkshead Lane, North Mymms, Hateld, Herts, UK

Contents
Culling for infertility remains the main reason for disposal of dairy cows, limiting productive lifespan. In extreme cases, ovulation is inhibited, preventing the possibility of conception. More often cows do conceive, but fail to remain pregnant owing to intrinsic problems in the embryo and or to a poorquality reproductive tract environment. Both aspects have a genetic component and are also inuenced by management practices aecting nutrition and health. The relative importance of these factors varies among heifers, rst-lactation and older cows. A common theme, however, is that an internal signalling system exists which reduces fertility when the cow is in an unsuitable metabolic state to sustain a pregnancy. This may be directly related to nutrient shortage caused by inadequate feed intake, or because available nutrients are being prioritized towards growth or milk production, away from reproduction. Evidence is presented for the involvement of the somatotrophic axis (GH, IGF1, insulin, IGFBP2) and leptin as key metabolic signalling molecules. Another emerging theme is the interaction between metabolism and disease that aects the fertility. Common examples include (i) calf diseases causing inadequate heifer growth and increased age at rst calving; (ii) poor peripartum energy status reducing the capacity of the uterus to involute and mount an eective immune response, thereby increasing the likelihood of endometritis; and (iii) development of mastitis after conception, a contributory factor to both early and late embryo mortality. Finally, recent evidence suggests that times of metabolic stress cause mitochondrial damage that also contributes to a reduction in longevity.

nisms involved and the interactions with disease that can inuence the fertility.

Causes of Reproductive Failure

Reproductive failure can have multiple causes, summarized briey here. Animals may not be inseminated because of a failure to ovulate (Peter et al. 2009) or to detect oestrus (Saint-Dizier and Chastant-Maillard 2012) or because of poor health, low milk yield, poor conformation or an unsuitable temperament. For cows that are undergoing oestrous cyclicity, fertilization rates generally exceed 90% (Diskin and Morris 2008), but many animals do not subsequently remain pregnant. Approximately, 40% of early embryos die in moderate producing dairy cows, mostly between 8 and 16 days post-insemination. This increases to 56% in higher yielding cows, with many embryos already showing abnormal development by day 7 (Diskin and Morris (2008). Several potential causes of early embryo mortality have been established. Firstly, oocyte quality may become compromised during periods of extreme negative energy balance (NEB), possibly due to excess accumulation of non-esteried fatty acids (NEFAs) (Van Hoeck et al. 2011), or toxic eects of elevated concentrations of ammonia and or urea associated with high protein diets (Leroy et al. 2008). Secondly, a variety of factors can inuence the ability of the reproductive tract to support an early pregnancy. Oestrogen and progesterone are key regulators of the tract environment, so anything that aects follicular and luteal development and their ability to secrete sucient hormone at the appropriate time can also inuence embryo development (Diskin and Morris 2008; Robinson et al. 2008). There are also metabolic inuences directly on tract secretions (Wathes et al. 2008a) and adverse eects of disease status (discussed below). Additional embryos die slightly later, mainly in the second month of gestation. These later losses, which occur at a frequency of 720% (Diskin and Morris 2008), are more frequent in higher yielding cows and many may be disease related. Numerous infectious agents have been associated with embryo mortality and abortion in cattle, including bovine virus diarrhoea virus (BVDV) and Neospora caninum, which are widespread in many dairy cow populations (Givens and Marley 2008). Another potential cause of embryo loss is the development of clinical mastitis (Hansen et al. 2004). Finally, some bulls and genotypes have been associated with fertilization failure and others with reduced embryo survival rates (Bulman 1979; Khatib et al. 2010). A recent study that transferred in vitro produced
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Introduction
Holstein dairy cows have an average productive lifespan of approximately three lactations or less (Hare et al. 2006). A cow only recoups her rearing costs during her second lactation, so protability improves with increased longevity, associated with a greater proportion of total lifetime spent in milk production (Jagannatha et al. 1998). Despite this, approximately 1520% of dairy cows are culled in their rst lactation, mainly due to poor fertility (Brickell and Wathes 2011). Understanding the underlying causes of poor fertility requires a holistic approach, encompassing aspects of reproductive biology, embryology, metabolism, immunology and genetics. This is being facilitated by recent advances in genomic technology. Use of gene expression arrays and association studies of single-nucleotide polymorphisms (SNPs) with fertility traits has helped to reveal which biological pathways are of key importance. This review considers why failure to conceive and remain pregnant continues to be the primary reason why so many dairy cows worldwide are culled at a relatively young age. The focus is on some of the underlying metabolic mecha-

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

Age-related Inuences on Fertility

For an individual cow to survive several lactations, she must remain consistently fertile. Age and lactation number both inuence fertility, so they need to be accounted for in models investigating reasons for reproductive failure. Heifer fertility is a key factor in determining age at rst calving (AFC). Animals with a delayed AFC have worse reproductive performance in the rst lactation and reduced longevity (Wathes et al. 2008a; Sakaguchi 2011). Respiratory diseases and diarrhoea are endemic within most populations of dairy calves (Johnson et al. 2011). These contribute to reduced growth rates and delayed puberty and prevent animals from reaching rearing targets (Brickell et al. 2009a). Kuhn et al. (2006) reported a conception rate of 56% in American Holstein heifers: conception rates were maximal at 1516 months of age, reducing by 13% in older animals (26 months). Dairy heifers should calve for the rst time at 2 years of age and 8290% mature body weight, so they must use nutrients for growth as well as for milk production during their rst lactation. They therefore dier metabolically to multiparous animals. Inadequate growth is a risk factor for maternal dystocia and can have detrimental inuences on fertility in the rst lactation (Ettema and Santos 2004). Conversely, late calving heifers are more likely to become overconditioned, also aecting calving ease. Perinatal mortality, a risk factor for subsequent poor fertility, is approximately two times more likely at rst calving (Brickell et al. 2009b). Fertility may be worse in rst-lactation cows in comparison with older animals (e.g. Wu et al. 2012), and they experience a higher incidence of delayed resumption of ovarian cyclicity (Wathes et al. 2007a). Although most older cows have achieved mature body size, they also have a greater capacity for milk production. Betahydroxybutyrate (BHB) concentrations are higher in early lactation, associated with the development of clinical or subclinical ketosis (Dueld et al. 1997; Wathes et al. 2007b). Higher lactation number and previous lactation milk yield both increase the risk of retained placenta (Fleischer et al. 2001). Older cows are also more likely to develop a persistent corpus luteum (Opsomer et al. 2000), and conception to a particular insemination declines once cows reach 5th parity (Inchaisri et al. 2010).

reached (e.g. Butler 2003; Westwood et al. 2002; Wathes et al. 2007a; Sakaguchi 2011). The most important determinant of BCS is dry matter intake (Hayirli et al. 2002). These variables are also inuenced by dry cow diet (Beever 2006) and the shape of the lactation curve. Some cows quickly reach a high peak milk yield associated with rapid BCS loss, whereas others achieve a high 305day milk yield through greater persistency of milk production.

Endocrine and Metabolic Factors

Metabolic and endocrine changes associated with nutrient shortage and endogenous body tissue mobilization have taken on signalling roles that prevent the cow establishing a pregnancy when conditions are suboptimal via inhibitory actions at the level of the brain, ovary or reproductive tract. Concentrations of NEFAs and BHB increase in early lactation, reecting the extent of adipose tissue mobilization and fatty acid oxidation, respectively (Bauman and Currie 1980). Glucose concentrations show a short-term decrease at this stage (Bell 1995), while urea concentrations may rise or fall, depending on the protein content of the diet and the degree of tissue mobilization (Laven et al. 2007). Such metabolite changes can inuence reproductive processes directly. In addition, they contribute to growth-related and post-partum changes in metabolic hormones that also aect the fertility.

IGF System and Fertility

Increased LH pulsatility during the peripubertal period promotes follicular development, leading ultimately to a sucient rise in oestradiol production to induce the rst pre-ovulatory LH surge (Day et al. 1987). This usually occurs at approximately 9 months, but only once the heifer possesses an adequate body size and or metabolic status to reproduce successfully (Rawlings et al. 2003; Taylor et al. 2004a). Numerous studies (reviewed by Velazquez et al. 2008) have provided evidence that IGF1 is an important metabolic mediator in the timing of puberty. The circulating IGF1 concentration is closely related to body weight during pre-pubertal growth, and faster-growing, well-fed animals attain puberty earlier (Macdonald et al. 2007). Systemic IGF1 concentrations in dairy heifers peak shortly before the onset of puberty, then gradually decline as growth rates decline (Brickell et al. 2009a; Fig. 1a). The start of lactation is also associated with profound changes in the somatotrophic axis. During metabolic shortages, the growth-promoting actions of GH mediated by IGF1 are curtailed and GH instead promotes tissue mobilization. This switch is primarily triggered by down-regulation of the liver-specic variant of the GH receptor (GHR1A) (Kobayashi et al. 1999; Fenwick et al. 2008a). This in turn is responsible for the pronounced decrease in circulating IGF1 concentrations, which begins before calving and typically reaches a nadir in the rst week post-partum (Taylor et al. 2004b; Fig. 1a). The actions of IGF1 are inuenced by concurrent changes in IGF-binding proteins (IGFBPs) (Jones and Clemmons 1995). The majority (>90%) of

Tissue Mobilization and Negative Energy Balance (NEB)

Modern dairy cows experience a period of nutrient shortage (NEB) in early lactation, as body reserves are mobilized to support milk output (Lucy 2001; Wathes et al. 2008b). Although the precise relationships are not always consistent among studies, fertility can be inuenced by the body condition score (BCS) before calving, the subsequent rate and extent of mobilization of body tissue, and the time at which the nadir in body weight is
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(a) Bodyweight (kg)

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0 (b) Bodyweight (kg)

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18 24 Age (months)

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(c)

The mechanisms by which IGF1 concentration can inuence fertility in cattle have been reviewed previously (e.g. Velazquez et al. 2008) and are summarized briey here. IGF1R are present on the pituitary gland, ovaries and reproductive tract. IGF1 can enhance LH secretion (Adam et al. 2000), increase follicular growth and oestradiol synthesis (Webb et al. 2004), promote uterine histotroph secretion (Wathes et al. 2008a) and increase the rate of early embryo development (Block 2007). In dairy heifers, reduced IGF1 concentrations at both 1 and 6 months of age were associated with delays in age at rst breeding and so an increase in AFC (Brickell et al. 2009c; Fig. 1c). First-lactation cows that experienced a delayed interval to rst ovulation post-partum had lower IGF1 concentrations than their peers at 6 months (Taylor et al. 2004a). Cows with a low postpartum nadir in their IGF1 concentration take longer to resume oestrous cycles following calving and are also less likely to conceive (Butler 2003; Taylor et al. 2004b; Patton et al. 2007). This nding is supported by studies in which bovine somatotrophin injection at the time of insemination improved conception rates in repeat breeder cows (Morales-Roura et al. 2001).

IGF1 (ng/ml)

IGF1 (ng/ml)

IGFBP2
Although all of the IGFBPs 16 are expressed in the ovary and or reproductive tract, our attention has focused particularly on IGFBP2, as this binding protein plays a key role in regulating IGF bioavailability in dierent tissues according to EB status. It is the second most abundant IGF-binding protein in the circulation, has high anity for both IGF1 and IGF2 and is generally considered to inhibit IGF activity (Jones and Clemmons 1995). Circulating concentrations of IGFBP2 increase after calving when the expression of the other IGFBPs is reduced (McGuire et al. 1995). In postpartum cows, hepatic IGFBP2 mRNA expression was positively correlated with circulating NEFA and BHB and negatively correlated with hepatic glycogen, blood glucose and IGF1 (Fenwick et al. 2008a). IGFBP2 is a key inhibitor of adipogenesis (Boney et al. 1994), and in humans, SNPs for IGFBP2 have been linked with diabetes, obesity and insulin resistance (Grarup et al. 2007). We have shown that IGFBP2 mRNA expression in ovarian granulosa cells and the oviduct decreased when cows were in severe NEB in early lactation, in contrast to the hepatic up-regulation at this time (Llewellyn et al. 2007; Fenwick et al. 2008b). We therefore investigated the associations between SNPs 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 CC genotypes of BP2_2 were signicantly older than heterozygotes at rst conception and subsequently produced higher 305-day milk yields than the heterozygotes, even though their lactations were shorter. The IGFBP2 SNP genotype was also associated with circulating glucose, insulin and BHB concentrations around calving. Associations of IGFBP2 SNPs with growth traits have also been reported in chickens and pigs (Lei et al. 2005; Mote and Rothschild 2006). Dierential regulation of IGFBP2 production by metabolic signals
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Fig. 1. (a) Changes in body weight (solid line) and circulating IGF1 (dashed line) in dairy heifers (n = 387) with age during their initial growth phase and around their rst and second calving (arrows). (b) The body weight and (c) the circulating IGF1 concentration of the heifers at 1 and 6 months of age according to their age at rst calving (AFC). Animals that were lighter and had lower IGF1 concentrations at these time points subsequently calved later, **p < 0.01:AFC <23.5 month (n = 56), 23.525.5 month (n = 122), 25.530 month (n = 163) and >30 month (n = 36)

hepatic-produced IGF1 normally circulates bound in a ternary complex with IGFBP3 and the acid-labile subunit (ALS). Their production is also regulated by the hepatic growth hormone receptor (GHR), so both decline in early lactation while the production of IGFBP2 rises (Fenwick et al. 2008a). These changes reduce the half-life of circulating IGF1. The ratio of IGF1 to IGF2 secreted also changes in the post-partum cow. Hepatic IGF2 production is not regulated by GH or aected by the stage of lactation (Fenwick et al. 2008a). Circulating IGF2 concentrations are thus maintained during periods of nutrient shortage when IGF1 concentrations are low. The local production of IGFBPs also regulates IGF activity. Most tissues express several IGFBPs that have variable anities for IGF1 and IGF2 (Jones and Clemmons 1995). Both IGF1 and IGF2 activate signalling through IGF1R, but the implications of a change in ligand for downstream signalling have not been addressed. It is thus hard to predict what the overall outcome will be in terms of the strength of activation of the IGF1R signalling pathway in a particular tissue in response to a particular circulating concentration of IGF1.

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in dierent tissues may thus control the availability of IGF1 and IGF2 to activate the IGF1R and so modulate the growth and reproduction with respect to nutrient availability.

Insulin
Insulin has an important inuence on nutrient partitioning. Decreasing insulin concentrations together with elevated placental lactogen levels in late gestation trigger adipose mobilization (Bell 1995). In the lactating multiparous cow, insulin concentrations are low postpartum and are negatively correlated with milk yield (Wathes et al. 2007b). Insulin infusion promotes an increase in hepatic GHR1A and IGF1 production after calving (Butler 2003). Relationships between the circulating insulin concentration and fertility outcomes have been demonstrated in beef cross heifers and lactating cows fed diets designed to enhance or reduce insulin secretion (Adamiak et al. 2005; Garnsworthy et al. 2009). However, for animals on more normal diets, we have failed to establish a relationship between insulin levels and fertility in either dairy heifers (Brickell et al. 2009c) or lactating cows (Wathes et al. 2007a). Insulin measurements are not as useful as IGF1 in predicting fertility outcomes for several reasons. They are strongly inuenced by time in relation to feeding, and both hypoand hyperinsulinaemia are associated with poor fertility outcomes. In addition, the main point of control in insulin signalling is at the level of the receptor rather than the circulating concentration. High lipid concentrations and acute infections can both cause peripheral insulin resistance and so reduce glucose uptake into nonessential 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, energy partitioning and adipose tissue deposition during both short- and long-term changes in nutritional state (Ingvartsen and Boisclair 2001). Circulating leptin concentrations in cattle are elevated pre-partum and at this stage are highly correlated with BCS; they decline at calving and then remain low even when the energy status has improved (Ingvartsen and Boisclair 2001; Wathes et al. 2007b). Peripartum leptin concentrations were signicantly higher in primiparous than in multiparous cows (Wathes et al. 2007b). Post-partum hypoleptinaemia may promote voluntary feed intake and contributes to peripheral insulin resistance (Ingvartsen and Boisclair 2001). With respect to possible direct eects on reproduction, leptin receptors are present in the bovine follicle and corpus luteum (Spicer 2001) and endometrium (Thorn et al. 2007). Leptin concentrations in cattle increase prior to puberty (Thorn et al. 2007) and may need to reach an adequate threshold level for the attainment of puberty (Cunningham et al. 1999). Two studies have shown associations between leptin polymorphisms with calving diculty and perinatal calf mortality, suggesting that problems in leptin signalling
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pre-partum may compromise placental and foetal development (Brickell et al. 2010; Giblin et al. 2010). A high leptin concentration before calving was a strong predictor of a delayed start to cyclicity and longer intervals to conception, but only in multiparous rather than in primiparous cows (Wathes et al. 2007a). Other studies have reported that low leptin concentrations after calving may contribute to long intervals to rst ovulation (Liefers et al. 2005). In ruminants, severe undernutrition is needed for leptin to inuence gonadotrophin secretion, so this eect may only be important in extreme circumstances (Zieba et al. 2005). Leptin can, however, aect the ovaries directly. Leptin promoted oocyte maturation in vitro, increasing both the fertilization rate and the proportion of embryos developing to the blastocyst stage (Boelhauve et al. 2005). While leptin alone had little eect on ovarian steroidogenesis, high leptin concentrations inhibited insulin or IGF1-stimulated oestradiol production in cultured granulosa cells (Spicer 2001), but had a synergistic eect with IGF1 to promote luteal progesterone production (Nicklin et al. 2007). Recent work on cancer cell lines has shown signicant interactions between intracellular actions of leptin and IGF (Saxena et al. 2008). Variations in circulating concentrations of leptin and IGFs associated with BCS and parity could therefore act synergistically to inuence reproductive tissues via cross-talk between their respective signalling pathways. We recently reported that several leptin gene polymorphisms were associated with fertility traits in dairy cows (Clempson et al. 2011a). In lactating cows, this included eects on days to conception and consequently calving interval. Previous studies in cattle have associated SNPs in leptin and should be its receptor with a variety of milk production traits and with dry matter intake (Liefers et al. 2005). Altered leptin activity could therefore have indirect eects on fertility via changes in energy balance status. Heifer fertility traits were, however, also aected in our study, including the number of services needed and AFC. This suggested that some actions on fertility are direct, possibly via the eects on the ovary and oocyte described above.

Mitochondria and Metabolic Rate

Surprisingly, little attention to date has focused on mitochondrial activity in the dairy cow. Mitochondria play a key role in intracellular energy production and are 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, causing damage to both proteins and DNA (Mammucari and Rizzuto 2010). Cells initially remove damaged organelles by autophagy, including breakdown of the mitochondria themselves (mitophagy), promoting the maintenance of a functional mitochondrial population and providing additional nutrients to the cell in times of shortage (Mammucari and Rizzuto 2010). When more extreme damage occurs, changes within the mitochondria instead promote apoptosis. The switch to an apoptotic pathway may be encouraged by growth factor withdrawal and increasing activity of p53, which senses

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intracellular stress signals including DNA damage, hypoxia and nutrient shortage (Mammucari and Rizzuto 2010). In our studies on the eects of severe NEB on tissues of 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 all tissues. 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 ample availability of long-chain fatty acids. PD4K is a mitochondrial enzyme that is up-regulated in response to an increased lipid supply, inactivating the pyruvate dehydrogenase complex and helping to conserve glucose by limiting the conversion of pyruvate to acetyl-CoA (Holness and Sugden 2003). TIEG1 is a transcription factor that can induce apoptosis via the mitochondrial pathway (Jin et al. 2007). TIEG1 is modied by the OGlcNAc pathway, which can reversibly alter protein activity according to the glucose availability (Alemu et al. 2011). We also investigated the associations between SNPs in two autosomal mitochondrial genes with fertility and milk production traits. TFAM encodes a histone-like protein essential for transcription and replication of mitochondrial DNA (Jiang et al. 2005). Uncoupling proteins (UCPs) transport protons across the inner mitochondrial membrane, contributing to the regulation of energy metabolism and the attenuation of ROS production (Echtay 2007). In beef cattle, polymorphisms in TFAM and UCP2 have been associated with subcutaneous fat depth, marbling and body weight (Jiang et al. 2005; Sherman et al. 2008). In the UK, Holstein-Friesian cows that were GG homozygotes for TFAM3 were less likely to conceive than heterozygotes, had a 24-day longer calving 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 GG homozygotes survived into a third lactation (Fig. 2a). The AA homozygotes also had slightly worse fertility. Only 33% of GG homozygotes for TFAM3 and 30% of AA homozygotes 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 no CC animals present. The heterozygotes had a reduced age at rst conception and a delayed return to cyclicity after rst calving when compared to the homozygotes. However, proportionately more GG than CG animals were culled before third calving (64 vs 37%, p < 0.05) (Clempson et al. 2011b; Fig. 2b). Single-nucleotide polymorphisms in both TFAM and UCP2 were also associated with growth traits in the heifers, but the relationship with survival only became evident once the animals had started to produce milk. It is an interesting possibility that modications to these proteins aect the ability of mitochondria to adapt to the changes in energy supply required at the start of lactation (Fig. 3). Excessive ROS damage at this time may then damage a variety of tissues, promoting disease and decreasing both fertility and longevity.

Metabolism and Disease

The transition from late pregnancy to early lactation is also associated with a compromised immune status (Cai et al. 1994; Mallard et al. 1998). Mounting an eective immune defence is energetically demanding (Fox et al. 2005), so during infection nutrients are targeted away from other body functions towards the immune system (Spurlock 1997). Both the level of impairment and the rate of recovery of immune capabilities post-partum are thus strongly inuenced by the extent of NEB around calving (Pyo ra la 2008; Wathes et al. 2009). This makes
(a) TFAM3

(b)

UCP2

Fig. 2. KaplanMeier analysis showing the proportion of animals surviving from birth through to the end of the second lactation according to (a) the TFAM3 single-nucleotide polymorphism (SNP) and (b) the UCP2 SNP (from Clempson et al. 2011b, with permission)

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Proteins imported from cytosol

Cellular stress (hypoxia, DNA damage, nutrient shortage) p53

NEB switches energy supply from glucose to NEFA

PDK4 mtDNA

Mitochondrial proteins mitophagy apoptosis ROS

Growth factor withdrawal eg reduced IGF1 TIEG1

mtDNA replica on

TFAM3

NADH NAD+

O2 H2O

UCP2 Electron transport chain heat

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. Damaged mitochondria are removed by mitophagy or apoptosis. The balance towards apoptosis is tipped by changes in whole-body metabolism reducing IGF1 concentrations, by increasing cellular stress acting through the p53 pathway and by up-regulation of the transcription factor TIEG1 inuencing gene expression. The ability of the remaining mitochondria to replicate and replace those which have been destroyed is inuenced by the genotype for TFAM3, while the UCP2 genotype may be important for the attenuation of ROS production, so limiting the initial damage

high-yielding cows in NEB susceptible to infection from the multiplicity of pathogenic organisms commonly present in the farm environment. Within 2 weeks after calving, 40% of cows develop metritis, whereas endometritis and or mastitis are present in approximately 15 and 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. Not only do ongoing infections disrupt the pre-ovulatory LH surge and inhibit normal follicular maturation (Sheldon et al. 2009), but an impaired uterine environment is almost certainly a major factor in repeat breeder cows that have a higher incidence of early embryonic death (Hill and Gilbert 2008). Mastitis is also associated with a reduction in pregnancy rate (Hansen et al. 2004; Schrick et al. 2011). There is a complex inter-relationship between acute infection, a predisposition to chronic inammation and a reduced capacity for tissue repair. This is of particular importance in the uterus, which must undergo extensive remodelling after calving before it is ready to establish another pregnancy. Using arrays to compare gene expression in endometrium of post-partum cows in severe or moderate NEB, we found dierential expression of 240 genes, nearly half of which were associated with immune or inammatory pathways (Wathes et al. 2009). There were also changes in the expression of genes associated with the IGF and insulin signalling pathways including IGFBP6, IGF1 and IGFALS which were all reduced. However, AHSG and PDK4, two genes implicated in insulin resistance, were more highly expressed in the endometrium of SNEB cows. As IGF1 has a positive eect on tissue repair mechanisms following injury (Mourkioti and Rosenthal 2005), our data support evidence that uterine involution and elimination of
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bacteria are delayed when cows are in NEB (Lewis 1997). This predisposes them to develop subclinical endometritis; aected animals have a markedly reduced rate of conception, and the proportion which fail to conceive at all also rises signicantly (Gilbert et al. 2005).

Conclusions
Infertility problems in dairy cattle are multifactorial and are associated with both genetics and management. Individual cow factors relating to the age and health of the animals, the amount of feed consumed and how it is utilized internally (nutrient partitioning) inuence their ability to conceive and remain pregnant. While each cow is bred with a genetic potential to achieve a certain level of milk production, this can only be realized if she is provided with a lifetime environment which enables her to full her potential; this requires her to remain fertile. Improvements in fertility can be achieved in the shortterm by identifying the main causes of infertility in a particular herd and adoption of optimized management strategies involving nutrition, reproductive management and animal health. A longer-term sustained improvement in fertility must also encompass appropriate genetic selection to identify females with high fertility traits using molecular genetic technologies. Acknowledgements
I am very grateful to my many excellent colleagues who have contributed to the research performed at the Royal Veterinary College and to DairyCo, Defra, Merial Animal Health Ltd., Volac International Ltd., BBSRC and the Wellcome Trust who helped to fund it.

Conicts of interest
The author does not have any conicts of interest to declare.

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