Leptin expression

Leptin is expressed mostly by all white adipose tissues (WAT), with lesser expression in brown fat (Cinti et al., Reference Cinti, Frederich, Zingaretti, De Matteis, Flier and Lowell1997), muscle, mammary gland, stomach, pituitary, placenta, ovary and liver. Stomach leptin (Bado et al., Reference Bado, Levasseur, Attoub, Kermorgant, Laigneau, Bortoluzzi, Moizo, Lehy, Guerre-Millo, Le Marchand Brustel and Lewin1998; Cinti et al., Reference Cinti, De Matteis, Picó, Ceresi, Obrador, Maffeis, Oliver and Palou2000) may target leptin receptors in the gut (Guilmeau et al., Reference Guilmeau, Buyse and Bado2004). Liver is the main leptin source in birds and fish (Johnson et al., Reference Johnson, Johnson and Londraville2000; Sato et al., Reference Sato, Nishida and Akiba2003), but mammalian liver does not express it. Expression is lower in mature muscle cells than in undifferentiated myoblasts (Solberg et al., Reference Solberg, Aas, Thoresen, Kase, Drevon, Rustan and Reseland2005) and intramuscular adipocytes may be a source in muscle (Ramsay and Richards, Reference Ramsay and Richards2005) as leptin stimulates proliferation of skeletal myoblasts (Yu et al., Reference Yu, Luo, Zhang, Wu, Zhang and Yang2008) but inhibits their differentiation. Gardan et al. (Reference Gardan, Gondret and Louveau2006) found that isolated porcine skeletal muscle adipocytes expressed leptin in amounts proportional to their growth (increasing diameter) in vitro, but always at lower levels than adipocytes isolated from porcine subcutaneous (s.c.) fat. Leptin is present in placental syncytiotrophoblasts that provide the interface between maternal and foetal bloods (Ashworth et al., Reference Ashworth, Hoggard, Thomas, Mercer, Wallace and Lea2000). The presence of leptin receptors also in placenta suggests autocrine or paracrine effects of leptin on processes like angiogenesis which leptin induces (Anagnostoulis et al., Reference Anagnostoulis, Karayiannakis, Lambropoulou, Efthimiadou, Polychronidis and Simopoulos2008) and which is essential to ensure an adequate nutrient supply for foetal growth and development.

Leptin in human and mouse milks (Houseknecht et al., Reference Houseknecht, McGuire, Portocarrero, McGuire and Beerman1997) suggests transfer from maternal blood or de novo synthesis in the mammary gland, or both. Epithelial cells are the source of milk leptin in man (Smith-Kirwin et al., Reference Smith-Kirwin, O’Connor, Johnston, De Lancey, Hassink and Funanage1998), mice (Aoki et al., Reference Aoki, Kawamura and Matsuda1999) and cows (Smith and Sheffield, Reference Smith and Sheffield2002) but Bonnet et al. (Reference Bonnet, Gourdou, Leroux, Chilliard and Djiane2002) detected it mostly in mammary adipocytes in early pregnancy. Leptin levels were 56% lower in day 10 bovine milk than in colostrum (Pinotti and Rosi, Reference Pinotti and Rosi2006) but correlated with fat in each (r = 0.90) and leptin-fortified formula milk increased the gut mucosal mitotic index in neonatal pigs (Wolinski et al., Reference Wolinski, Biernat, Guilloteau, Westrom and Zabielski2003). Colostral leptin might speed gut maturation – but sow milk leptin level did not affect litter size or birth weight or weaning weight of pigs (Whitley et al., Reference Whitley, O’Brien, Quinn, Keisler, Walker and Brown2009).

Leptin among animal species

Leptin is well characterised in man, laboratory rodents, farm animals and other placental land mammals as well as marsupials and fish and seals and whales. Its presence in birds is broadly accepted despite disagreement about the identity of the avian gene (see later). Ji et al. (Reference Ji, Willis, Scott and Spurlock1998) and Kawakita et al. (Reference Kawakita, Abe and Hodate1999) cloned the bovine leptin gene and found 82% to 88% homology with human and mouse. The pig leptin gene shares 85%, 88% and 92% homology with mouse, man and cow, respectively (Ramsay et al., Reference Ramsay, Yan and Morrison1998) while bovine and ovine leptins differ by only two aa (Gertler et al., Reference Gertler, Simmons and Keisler1998). The gene sequence reported for chicken (Taouis et al., Reference Taouis, Chen, Davidaud, Dupont, Derouet and Simon1998; Ashwell et al., Reference Ashwell, Czerwinski, Brocht and McMurtry1999) was disputed by Friedman-Einat et al. (Reference Friedman-Einat, Boswell, Horev, Girishvarma, Dunn Talbot and Sharp1999) and by Burt (Reference Burt2004) but Richards et al. (Reference Richards, Caperna, Elsasser, Ashwell and McMurtry2000) used antibody to 15-aa of pig leptin to identify leptin protein in chicken plasma, fat and liver while Neglia et al. (Reference Neglia, Arcamone, Gargiulo and de Girolamo2008) showed it in chicken gut. However, Ninov et al. (Reference Ninov, Ledur, Alves, Fernandes do Rosário, Nones and Coutinho2008) could not amplify any of three primers deduced from Genbank AFO12727 or another from Dridi et al. (Reference Dridi, Buyse, Decuypere and Taouis2005a). Low nucleotide sequence homology between species need not mean radically different proteins, e.g. pufferfish leptin has <25% homology with mammalian leptin but similar tertiary structure (Yacobovitz et al., Reference Yacobovitz, Solomon, Gusakovsky, Levavi-Sivan and Gertler2008). On the other hand, recombinant chicken leptin (re-ch.lep) and human leptin (re-h.lep) similarly affected feed intake by chickens while mouse leptin had no effect (Bungo et al., Reference Bungo, Shimojo, Masuda, Tachibanab, Tanaka, Sugahara and Furuse1999) despite high aa-sequence homology with chicken (Ashwell et al., Reference Ashwell, Czerwinski, Brocht and McMurtry1999). Turkey leptin gene was partly sequenced by Ashwell et al. (Reference Ashwell, Czerwinski and McMurtry1998) while Dai et al. (Reference Dai, Long, Zhang, Zhang and Wu2007) cloned it from duck and showed 96%, 92% and 84% aa-sequence homology with rat, pig and human. Interestingly, leptin has been shown in a migratory bird (dunlin; Kochan et al., Reference Kochan, Karbowska and Meissner2006) with expression traced to liver and adipose tissue while, in fish, liver was also shown as the major source (Johnson et al., Reference Johnson, Johnson and Londraville2000).

Secretory characteristics

Leptin secretion is pulsatile in man (Licinio et al., Reference Licinio, Negrao, Mantzoros, Kaklamani, Wong, Bongiorno, Negro, Mulla, Veldhuis, Cearnal, Flier and Gold1998) and rat (Bagnasco et al., Reference Bagnasco, Kalra and Kalra2002). Wylie et al. (Reference Wylie, Mackey, Devlin, Bjourson and Diskin2001) found 8 to 10 pulses per 10 h in heifers while Tokuda et al. (Reference Tokuda, Matsui and Yano2000) noted almost 5 per day in ewes. Secretion in rams was described as episodic rather than pulsatile (Blache et al., Reference Blache, Tellam, Chagas, Blackberry, Vercoe and Martin2000). Fasting reduced pulse frequency in ewes (Daniel et al., Reference Daniel, Whitlock, Baker, Steele, Morrison, Keisler and Sartin2002) but 12 days of feed restriction (0.4 M v. 2 M) did not alter the pulsatility in beef heifers (Wylie et al. (Reference Wylie, Mackey, Devlin, Bjourson and Diskin2001). Barb et al. (Reference Barb, Barrett, Kraeling and Rampacek2001a) noted pulsatile secretion in pigs, and restriction of gilts reduced pulse frequency from 2.7 to 1.8 in 4 h (Whisnant and Harrell, Reference Whisnant and Harrell2002). Some reports indicate higher nocturnal levels in some species. Expression is stimulated by oestrogen (Shimizu et al., Reference Shimizu, Shimomura, Nakanishi, Futawateri, Ohtani, Sato and Mori1997) and inhibited by testosterone (Wabitsch et al., Reference Wabitsch, Blum, Muche, Braun, Hube, Rascher, Heinze, Teller and Hauner1997) so that secretion is sexually dimorphic with pulse height and total output higher in women (2 ×) than men (Licinio et al., Reference Licinio, Negrao, Mantzoros, Kaklamani, Wong, Bongiorno, Negro, Mulla, Veldhuis, Cearnal, Flier and Gold1998) even at equal fat content (Saad et al., Reference Saad, Damani, Gingerich, Riad-Gabriel, Khan, Boyadjian, Jinagouda, El-Tawil, Rude and Kamdar1997). Levels are higher in ewes than wethers or rams (Blache et al., Reference Blache, Tellam, Chagas, Blackberry, Vercoe and Martin2000) and in heifers than steers despite lighter carcases (Brandt et al., Reference Brandt, Keisler, Meyer, Schmidt and Berg2007). Gonadectomy reduced leptin secretion in both male and female rats (Bagnasco et al., Reference Bagnasco, Kalra and Kalra2002) but did not disturb the sexual dimorphism. Fat distribution does not seem to dictate the dimorphism in leptin concentrations (Rosenbaum et al., Reference Rosenbaum, Pietrobelli, Vasselli, Heymsfield and Leibel2001) even though leptin is more strongly linked to the s.c. fat that accumulates more in women.

The leptin receptor and intracellular signalling

Many membrane-embedded hormone receptors use kinases in their intra-cellular signalling actions. As a class I cytokine receptor, ObR has no intrinsic kinase but instead uses Janus kinase (JAK) to phosphorylate STAT3, one of the family of Signal Transducers and Activators of Transcription, and an essential molecular component of the leptin signalling pathway (Buettner et al., Reference Buettner, Pocai, Muse, Etgen, Myers and Rossetti2006). The leptin intracellular signalling pathway intersects with those of other hormones (e.g. insulin) in muscle (Strat et al., Reference Strat, Kokta, Dodson, Gertler, Wu and Hill2005) and other tissues (e.g. liver), resulting in interactions between leptin and insulin (the universal anabolic hormone), which may determine how and why individual tissues differently metabolise glucose and fatty acids. Such interactions may have particular significance in ruminants in which fatty acid supply is always generous and in which monogastric-like hypoglycaemic : hyperglycaemic perturbations do not naturally occur.

Leptin receptor in livestock species

Dyer et al. (Reference Dyer, Simmons, Matteri and Keisler1997) screened hypothalamus, anterior pituitary and WAT in ovariectomised ewes and found ObR mRNA in each. In situ hybridisation localised it to ventromedial and arcuate nuclei in the hypothalamus, with more in feed-restricted ewes than in well-fed ewes. Chelikani et al. (Reference Chelikani, Glimm and Kennelly2003) found it in bovine WAT, liver, abomasum, mammary parenchyma, small intestine and brain with ObRa in the liver and pituitary. In prepubertal heifers, ObRb accounted for 40% of all ObR in the hypothalamus but little (<3%) elsewhere, while ObRa accounted for nearly all ObR in peripheral tissues but only 19% of hypothalamic ObR (Thorn et al., Reference Thorn, Meyer, Van Amburgh and Boisclair2007). Ren et al. (Reference Ren, Wegner, Bellmann, Brockmann, Schneider, Teuscher and Ender2002) found no difference in hypothalamic ObR expression in dairy-type Holstein or beef-type Charolais bulls fed a common diet to slaughter at 18 months but the Holsteins had higher circulating leptin and more omental and renal fat, consistent with their production-type. Actually, ObRb, ObRa and ObRc are all strongly expressed in bovine intermuscular, renal and s.c. fat (Kawachi et al., Reference Kawachi, Yang, Hamano, Matsui, Smith and Yano2007) suggesting that these tissues respond to leptin in addition to secreting it while ObRa was found also in lactating ewe mammary gland (Laud et al., Reference Laud, Gourdou, Belair, Keisler and Djiane1999). Ogasawara et al. (Reference Ogasawara, Ohwada, Nagai, Taketa, Matsuzaki, Tanaka, Watanabe, Aso and Yamaguchi2008) identified ObRb in the anterior pituitaries of Holstein bull calves, with a similar distribution of somatotrophs (60%) and gonadotrophs (16%) to that seen in mature ewes (Iqbal et al., Reference Iqbal, Pompolo, Considine and Clarke2000). Lin et al. (Reference Lin, Barb, Matteri, Kraeling, Chen, Meinersmann and Rampacek2000) found ObRb mRNA in the hypothalamus, cerebral cortex, pituitary and other tissues of 3-month old pigs suggesting leptin pleitropy in pigs, as in other mammalian species.

The existence of binding sites of different affinities for a hormone is common. Margetic et al. (Reference Margetic, Gazzola, Pegg and Hill2002b) used classical (Scatchard) analysis to characterise leptin binding to crude bovine kidney cell membrane preparations over a range of leptin concentrations from physiological (∼1 ng/ml) to excessively supra-physiological (∼150 ng/ml), and found two binding sites – one of high affinity and low abundance; the other of low affinity but almost ×100 greater abundance – suggesting that the effects of acute changes in leptin concentration (endogenously or exogenously) might be muted by the natural buffering of leptin, even at high doses. It is clear that the manner and extent of leptin binding to receptors and carrier proteins (in blood and at the tissues) determines the degree of intracellular effects. Accordingly, leptin pharmacokinetics are of significant interest to the genesis and treatment of human obesity but also to manipulative applications in farm livestock (Strat et al., Reference Strat, Kokta, Dodson, Gertler, Wu and Hill2005).

Mammalian leptins influenced feed intake, reproduction, energy metabolism and immune function in birds only in some studies. Bungo et al. (Reference Bungo, Shimojo, Masuda, Tachibanab, Tanaka, Sugahara and Furuse1999) injected mouse leptin i.c.v. into hens and found no reduction in intake, whereas Dridi et al. (Reference Dridi, Swennen, Decuypere and Buyse2005b) infused re-ch.lep for 6 h into 3-week old broilers and reduced feed intake and ch-ObR expression. Hen et al. (Reference Hen, Yosefi, Ronin, Einat, Rosenblum, Denver and Friedman-Einat2008) transfected ch-ObR into human embryo kidney cells and developed a bioassay that could distinguish pre-partum from post-partum (PP) cows. Ironically, the same assay could not distinguish lean from fat birds. Proteins of 1148 and 1147-aa with ∼50% homology to mammalian ObR have been shown in hens (Horev et al., Reference Horev, Einat, Aharoni, Eshdat and Friedman-Einat2000) and turkeys (Richards and Poch, Reference Richards and Poch2003) and exon 20 of avian ObR is spliced, giving a short isoform as in mammals (Liu et al., Reference Liu, Dunn, Sharp and Boswell2007b). Ohkubo et al. (Reference Ohkubo, Nishio, Tsurudome, Ito and Ito2007) used a monoclonal antibody to part of ch-ObR to map it and found the short isoform in only a few tissues but the long isoform expressed more widely (mostly brain). These avian receptors use JAK-STAT (Adachi et al., Reference Adachi, Takemoto, Bungo and Ohkubo2008) and are expressed in ovary, brain and pituitary (Paczoska-Eliasiewicz et al., Reference Paczoska-Eliasiewicz, Gertler, Proszkowiec, Proudman, Hrabia, Sechman, Mika, Jacek, Cassy, Raver and Rzasa2003).

Leptin as an indicator of fat stores

Correlations initially found between leptin and body fat in man and mice (Frederich et al., Reference Frederich, Hamann, Anderson, Lollmann, Lowell and Flier1995; Maffei et al., Reference Maffei, Halaas, Ravussin, Pratley, Lee, Zhang, Fei, Kim, Lallone, Ranganathan, Kern and Friedman1995) triggered interest in leptin as a fat marker in farm species and correlations with body condition and fat mass have been widely shown. When EI and EE are equal, leptin concentrations reflect the total amount of triglyceride (Barb et al., Reference Barb, Hausman and Ramsay2006). Early studies in cattle and sheep used a radioimmunoassay (Linco multi-species RIA) based on anti-recombinant human leptin but Delavaud et al. (Reference Delavaud, Bocquier, Chilliard, Keisler, Gertler and Kann2000), Ehrhardt et al. (Reference Ehrhardt, Slepetsis, Siegal-Willott, Van Amburgh, Bell and Boisclair2000) and Blache et al. (Reference Blache, Tellam, Chagas, Blackberry, Vercoe and Martin2000) raised antibodies to recombinant ovine or bovine leptins and developed ruminant-specific RIAs. Tokuda et al. (Reference Tokuda, Delavaud and Chilliard2003) reported a poor correlation (r = 0.41) between the non-specific and specific assays.

Leptin and appetite

Leptin is an anorexigenic agent shown to regulate food intake (Houseknecht and Portocarrero, Reference Houseknecht and Portocarrero1998). Its effects on intake are centrally mediated and rapid (Henry et al. (Reference Henry, Goding, Alexander, Tilbrook, Canny, Dunshea, Rao, Mansell and Clarke1999) injected leptin i.c.v. into ovariectomised ewes and reduced food intake significantly within 24 h). However, leptin’s central actions are rate-limited by BBB transport (Caro et al., Reference Caro, Kolaczynski, Nyce, Ohannesian, Opentanova, Goldman, Lynn, Zhang, Sinha and Considine1996) and leptin is just one of several agents affecting fat level. Links between leptin and fatness are influenced by nutrition but, conversely, the impact of nutritional change on leptin is affected by the degree of fatness and also by age (Ishii et al., Reference Ishii, Shibasaki, Murakami, Shima and Wakabayashi2000).

Undomesticated animals mostly do not over-consume food. Food scarcity undoubtedly is a greater threat and the main purpose of leptin may be to ensure adequate fat storage for survival when food is unavailable for whatever reason (e.g. food shortage, hibernation or migration). Cold-water mammals accumulate very large amounts of fat as thermal insulation, an energy source and a substrate for milk synthesis but Ortiz et al. (Reference Ortiz, Houser, Wade and Ortiz2003) saw no change in leptin during a 2 to 3 months post-weaning fast in Northern elephant seals, yet Guilherme et al. (Reference Guilherme, Bianchini, Martinez, Robaldo and Colares2004) reported a fall in leptin in adult seals leaving the sea to moult or calf. Some bird species interrupt migration to feed and replete their fat stores and it would seem important, or desirable, that they ‘know’ the size of their fat reserves as depletion of these reserves eventually leads to loss of body (muscle) protein. Leptin may be the marker but there is no evidence, yet, to link leptin levels to migratory success in birds (Kochan et al., Reference Kochan, Karbowska and Meissner2006). Leptin may also induce the hypothermia that develops in Barnacle geese days before migration, reducing energy use and conserving it as fat (Butler and Woakes, Reference Butler and Woakes2001). Hibernation also impacts energy budgets, as epitomised by 20% lower energy use in Siberian hamsters during periods of winter torpor of up to 8 h/day (Ruf and Heldmaier, Reference Ruf and Heldmaier1992), with hamsters that enter torpor nightly having the lowest leptin level. Leptin infusion prevented torpor in hamsters that most strongly exhibited it (Freeman et al., Reference Freeman, Lewis, Kauffman, Blum and Dark2004) and, so, low leptin may be linked to the associated suspension of reproductive activity.

Farm livestock other than breeding stock live short and closely managed lives to slaughter, or longer but equally closely managed lives producing milk or eggs. Obesity is rarely a concern, even amongst long-lived breeding stock, but poor feed management can allow excessive fat accumulation. Livestock are generally fed adequately to sustain their production potential but feed shortages occasionally occur. Amstalden et al. (Reference Amstalden, Garcia, Williams, Stanko, Nizielski, Morrison, Keisler and Williams2000) noted a fall in leptin mRNA and plasma leptin in feed-restricted cattle and sheep although Wylie et al. (Reference Wylie, Devlin and Bjourson2002) found no change in plasma leptin when wethers previously held at maintenance intake for more than 20 days were then acutely fasted. Moreover, levels remained unchanged during a 14-day staged return to M intake and increased significantly only after 7 days at 2 M. Thomas et al. (Reference Thomas, Wallace, Aitken, Mercer, Trayhurn and Hoggard2001) noted that leptin levels increased in adult rams within 5 days after increasing their feed allowance. Delavaud et al. (Reference Delavaud, Ferlay, Faulconnier, Bocquier, Kann and Chilliard2002) fed fat Charolais cows, fat Holstein cows and lean Holsteins at 1.3 or 0.6 M for 3 weeks and found initial leptin 70% lower in lean Holsteins than in fat Holsteins. Restriction depressed leptin by ∼25% but leptin levels fell post-prandially in well-fed cows and increased in underfed cows. The PP period in dairy cows is an exceptional time in which energy demand for milk synthesis can vastly outstrip supply resulting in a severely negative energy balance (NEB) and fat mobilisation. The accompanying fall in circulating leptin levels may be important to allowing feed intake to progressively increase (Kadokawa and Martin, Reference Kadokawa and Martin2006).

In neonatal lambs fed to gain at 148 or 337 g/day, leptin increased within 6 days in well-fed lambs and then continued to rise but were unchanged in slower growing lambs despite increasing fatness (Ehrhardt et al., Reference Ehrhardt, Greenwood, Bell and Boislair2003). Variation in leptin in sheep was considered by Delavaud et al. (Reference Delavaud, Bocquier, Chilliard, Keisler, Gertler and Kann2000) to be more attributable to body fatness (35%) than to nutritional status (17%) while intake, liveweight and backfat together accounted for 40% (18%+17%+5%) of the variation in leptin in dairy heifers (Chelikani et al., Reference Chelikani, Ambrose, Keisler and Kennelly2009). Growth rate variations between pig breeds, and within and between litters, are due mostly to variation in feed intake and leptin’s association with intake regulation may find practical use in improving pig production. Giving leptin i.v. to pigs reduced feed intake (Ajuwon et al., Reference Ajuwon, Kuske, Anderson, Hancock, Houseknecht, Adeola and Spurlock2003) as anticipated, but leptin levels in pigs are more strongly correlated with fatness than intake (Cameron et al., Reference Cameron, Penman and McCullough2000). Liver leptin expression and plasma leptin were higher in broilers selected for high abdominal fat (Dridi et al., Reference Dridi, Buyse, Decuypere and Taouis2005a) but infusion of re-ch.lep into 3-month old birds halved intake. Kuo et al. (Reference Kuo, Cline, Werner, Siegel and Denbow2005) infused re-h.lep into hens selected, long-term, for high or low 8-week weight, but reduced intake only in the lighter birds, suggesting a change in their leptin responsiveness and a potential target for genetic improvement. Reports of leptin’s associations with food intake in fish are mixed. Johnson et al. (Reference Johnson, Johnson and Londraville2000) found 3 × higher leptin in fed than fasted fish but a 6-week fast did not affect expression in carp (Huising et al., Reference Huising, Geven, Kruiswijk, Nabuurs, Stolte, Spanings, Verburg-van Kemenade and Flik2006) while injection of leptin reduced food intake by goldfish (De Pedro et al., Reference De Pedro, Martinez-Alvarez and Delgado2006). Clearly, a role for leptin in regulating appetite and energy use in fish could have important implications for profitability and for reducing the environmental impact of aquaculture.

Leptin and meat quality characteristics

In the context of this review, the most significant fats are the otherwise mundane triglycerides that constitute most of the WAT, protecting bones, muscles and organs against injury and providing thermal insulation. Importantly, fat depots accumulate and later mobilise triglyceride, assuring the availability of fatty acids during periods of NEB, whether caused by food scarcity or increased production demand or both.

Triglycerides comprise most of the i.m. and marbling fat in meat, contributing to cooking quality and eating quality (Hausman et al., Reference Hausman, Dodson, Ajuwon, Azain, Barnes, Guan, Jiang, Poulos, Sainz, Smith, Spurlock, Novakofski, Fernyhough and Bergen2009). Minton et al. (Reference Minton, Bindel, Drouillard, Titgemeyer, Grieger and Hill1998) associated leptin with marbling score in beef cattle and showed a positive relationship with L. dorsi area (r = 0.32), predating reported correlations between L. dorsi area and marbling score and s.c. fat in cattle (Bertrand et al., Reference Bertrand, Green, Herring and Moser2001). Geary et al. (Reference Geary, McFadin, MacNeil, Grings, Short, Funston and Keisler2003) found leptin 1 to 3 days before slaughter similarly correlated with marbling in 14 to 17 months crossbred steers and heifers but only weakly or negatively related to L. dorsi area (r = 0.12 in Angus × Charolais; r = −0.45, others) while Brandt et al. (Reference Brandt, Keisler, Meyer, Schmidt and Berg2007) found leptin only weakly related to marbling score (r = 0.28) in beef heifers and steers. In steers with 0%, 50% and 75% Wagyu genetics, leptin levels were unrelated to s.c. fat depth in all groups but correlated positively with percentage of fat in pars costalis and Musculus longissimus in zero-Wagyu steers both at 16 weeks pre-slaughter (r = 0.69; 0.59) and at 4 weeks pre-slaughter (r = 0.52; 0.51). The correlations disappeared in 50% Wagyu steers and became strongly negative (r = −0.65; −0.60) in 75% Wagyu steers. Daix et al. (Reference Daix, Pirotte, Bister, Wergifosse, Cuvelier, Cabaraux, Kirschvink, Istasse and Paquay2008) compared carcase characteristics, blood leptin level and leptin expression in s.c. fat of Belgian Blue, Angus and Limousin bulls. Fat content was 10.2% in Belgian Blue and 23.6% in Angus with Limousin intermediate. The more highly muscled Belgian Blues had the lowest leptin expression but plasma leptin level did not differ from the other breeds until adjusted to common carcase fat, when it became significantly higher. Low marbling in Belgian Blues is in line with the inverse relationship of leptin and marbling seen in cattle with high Wagyu genetics (Wegner et al., Reference Wegner, Huff, Xie, Schneider, Teuscher, Mir, Mir, Kazala, Weselake and Ender2001). Diets with low vitamin A content also are associated with higher marbling and better carcase grading in cattle (Oka et al., Reference Oka, Maruo, Miki, Yamasaki and Saito1998). Intriguingly, plasma vitamin A and leptin levels were inversely related in Wagyu (Yang et al., Reference Yang, Kawachi, Khan, Lee, Kim, Ha Jong, Lee, Lee, Ki, Kim, Sakaguchi, Maruyama and Yano2008) yet Tokuda et al. (Reference Tokuda, Kono and Fujihara2001) supplemented Japanese Black steers with vitamin A from birth to 20 months with no effect on circulating leptin concentrations. Gorocica-Buenfil et al. (Reference Gorocica-Buenfil, Fluharty, Bohn, Schwartz and Loerch2007) restricted vitamin A intake by Holstein steers for 112 days but did not affect intake, gain, carcase weight or backfat depth. However, i.m. fat percentage in L. dorsi increased by 33% after 243 days of restriction, and i.m. fat cell number (but not size) also increased, but effects on marbling were inconsistent (Gorocica-Buenfil et al., Reference Gorocica-Buenfil, Fluharty and Loerch2008).

Leptin and puberty

Exogenous leptin restored gonadotropin secretion in aleptinaemic ob mice (Chehab et al., Reference Chehab, Lim and Lu1996), reversing the sterility of both females and males. It also caused vaginal opening and it advanced oestrus in normal, prepubertal females (Ahima et al., Reference Ahima, Dushay, Flier, Prabakaran and Flier1997; Chehab et al., Reference Chehab, Mounzih, Lu and Lim1997). Humans with the exceptionally rare condition of congenital aleptinaemia remain prepubertal into adulthood but Farooqi et al. (Reference Farooqi, Jebb, Langmack, Cheetham, Prentice, Hughes, McCamish and O’Rahilly1999) treated a congenitally aleptinaemic 9-year old female with no overt sexual development with leptin and raised gonadotropin levels, allowing puberty to proceed.

Brody (Reference Brody1945) linked puberty in cows to an inflection of the growth curve coincident with the start of sustained fat gain. Ewes also must attain a fat threshold for puberty to proceed (Pittroff et al., Reference Pittroff, Dahm, Blanc, Keisler and Cartwright2008). Changes in leptin level and leptin mRNA expression were associated with puberty onset in cattle (Garcia et al., Reference Garcia, Amstalden, Williams, Stanko, Morrison, Keisler, Nizielski and Williams2002) and pigs (Barb and Kraeling, Reference Barb and Kraeling2004). Williams et al. (Reference Williams, Amstalden, Garcia, Stanko, Nizielski, Morrison and Kiesler2002) noted leptin increased in heifers going through puberty but Chelikani et al. (Reference Chelikani, Ambrose, Keisler and Kennelly2009) saw no distinct peripubertal leptin rise in heifers fed to gain at 0.5, 0.8 or 1.1 kg/day. Block et al. (Reference Block, Smith, Ehrhardt, Diaz, Rhoads, Van Amburgh and Boisclair2003) compared heifers entering puberty early or late (286 days v. 414 days) and found early heifers lighter, leaner and lower in leptin (2.6 ng/ml v. 4.3 ng/ml) suggesting it is not a pre-requisite for puberty but Zieba et al. (Reference Zieba, Amstalden and Williams2005) and Zhang et al. (Reference Zhang, Blache, Blackberry and Martin2005) regarded it as at least permissive to puberty in heifers and sheep. Cunningham et al. (Reference Cunningham, Clifton and Steiner1999) and some others have described leptin as a ‘metabolic gate’ to initiating reproductive activity in the hypothalamus, the centre of appetite regulation and location of most mammalian leptin receptors.

Paczoska-Eliasiewicz et al. (Reference Paczoska-Eliasiewicz, Proszkowiec-Weglarz, Proudman, Jacek, Mika, Sechman, Rzasa and Gertler2006) injected 11-week pre-pubertal pullets daily for several weeks with four doses of re-ch.lep. The highest dose advanced first lay (116.3 days v. 121.3 days). In another trial, the highest dose moved puberty from 124.4 to 118.4 days in fed birds and from 131.5 to 125.7 days in restricted birds and promoted follicle development in fully fed birds. Overall, the data suggests leptin may advance puberty in some farm species while only allowing it to proceed in others when energy reserves (indicated by leptin) are adequate. This is an important distinction.

Leptin in pregnancy

ObR mRNA in immature and mature bovine oocytes and embryos suggested a role for leptin in implantation and embryo development (Madeja et al., Reference Madeja, Warzych, Peippo, Lechniak and Switonski2009b) as well as in ovary development and steroidogenesis (Henson and Castracane, Reference Henson and Castracane2006). Maternal leptin drives conceptus growth and placental angiogenesis but placental leptin may also be involved. Ruminant placenta was thought to produce little leptin until Ehrhardt et al. (Reference Ehrhardt, Slepetsis, Bell and Boisclair2001) fed monotocous ewes to allow growth of the gravid uterus but avoid non-gravid growth, and found leptin levels higher than in non-pregnant ewes similarly fed to avoid gain, suggesting the placenta does indeed augment maternal leptin supply.

Leptin given to rabbit does for 1-week before inducing ovulation did not affect offspring number or weight but did increase the proportion of pregnant females and numbers of live offspring (Sirotkin et al., Reference Sirotkin, Rafay and Kotwica2009). Malik et al. (Reference Malik, Carter, Murray, Scaramuzzi, Wilson and Stock2001) injected leptin into female ob mice to restore levels to those of sexually mature ‘wild-type’ mice, and then mated leptin-treated ob females with either wild-type males or with ob males treated with leptin. Pregnant ob females carried to term only if leptin injections were maintained to at least day 7 post coitus (pc) but, in a similar study (Mounzih et al., Reference Mounzih, Qiu, Ewart-Toland and Chehab1998), leptin-treated ob females carried to term even if treatment was discontinued on day 1 pc suggesting that leptin is not essential for implantation or for any stage of gestation. However, the latter study used human leptin at 10 times the dose of Malik et al. (Reference Malik, Carter, Murray, Scaramuzzi, Wilson and Stock2001). Such differences matter when interpreting leptin’s effects.

Leptin and the hypothalamo–pituitary axis

Nutrition underpins animal reproduction and if leptin is a metabolic gate within the neuroendocrine axes, then low leptin in underfed animals and those in severe NEB (e.g. high-yielding early PP dairy cows) keeps the gate shut, ensuring that an apparently unsupportable pregnancy is prevented. Pubertal onset in cattle is accompanied by higher secretion of gonadotropin releasing hormone (GnRH) that increases the essential LH pulses (Zieba et al., Reference Zieba, Amstalden and Williams2005). Fasting suppresses FSH levels and pulsatile LH secretion with a temporary loss of fertility. The effects are linked to a fall in GnRH and are reversed by exogenous GnRH (Kile et al., Reference Kile, Alexander, Moss, Hallford and Nett1991). Re-feeding also restores normal LH secretion and relieves infertility induced by fasting (Cameron and Nosbisch, Reference Cameron and Nosbisch1991). Furthermore, exogenous leptin accelerated follicular growth in ob mice treated with a GnRH antagonist to prevent GnRH release (Barkan et al., Reference Barkan, Hurgin and Dekel2005) suggesting that leptin can induce ovulation independently of LH. Yu et al. (Reference Yu, Kimura, Walczewska, Karanth and McCann1997) showed it to be almost as effective as the natural secretagogue in stimulating GnRH release from arcuate nuclei, even at low dose. Leptin fell sharply in heifers during a 2-day fast near puberty, alongside lower LH pulse frequency (Amstalden et al., Reference Amstalden, Garcia, Williams, Stanko, Nizielski, Morrison, Keisler and Williams2000) while pulse frequency was not affected in mature cows in good condition and leptin levels correlated with pulse frequency (r = 0.731) and amplitude (r = 0.528) in PP Holsteins (Kadokawa et al., Reference Kadokawa, Blache and Martin2006), when EB was strongly negative (−100 MJ/day).

When administered i.c.v., leptin induced the hypersecretion of LH in fasted cows (Williams et al., Reference Williams, Amstalden, Garcia, Stanko, Nizielski, Morrison and Kiesler2002) but not in well-fed cows while i.c.v. infusion into ewes restricted for 6 months (23 kg below controls) restored LH (Henry et al., Reference Henry, Goding, Tilbrook, Dunshea and Clarke2001). Yet Maciel et al. (Reference Maciel, Zieba, Amstalden, Keisler, Neves and Williams2004) infused ovine leptin into prepubertal heifers for 40 days and raised plasma leptin by 7 times but did not affect LH pulses, or mean LH, or induce puberty. Morrison et al. (Reference Morrison, Daniel, Holmberg, Djiane, Raver, Gertler and Keisler2001) found lower LH pulse frequency in restricted lambs than fed lambs and this did not change with i.c.v. leptin infusion. However, Wójcik-Gładysz et al. (Reference Wójcik-Gładysz, Wańkowska, Misztal, Romanowicz and Polkowska2009) infused leptin i.c.v. into acutely undernourished prepubertal lambs and increased LH pulsatility. In pigs, i.c.v. leptin injection (10 to 100 μg) into prepubertal gilts did not affect serum LH (Barb et al., Reference Barb, Hausman and Czaja2005) but pig pituitary cells treated with GnRH and >1 nM leptin (alone or combined) had increased LH secretion suggesting that leptin affects both hypothalamic GnRH release and pituitary LH secretion directly.

Leptin and mammalian fertility

Fertility can be defined so as to reflect the success of parts of the reproductive cycle or to give a holistic view from the development of spermatozoa and oocytes, through ovulation and fertilisation, to established pregnancy and, assuming embryo survival, to parturition. Nutrition supports these processes, directly or indirectly, and fertility is affected by extremes of EB. Obese women often are amenorrheic with associated reproductive failure (Gosman et al., Reference Gosman, Katcher and Legro2006) but women with only minimal body fat levels may be similarly affected (De Souza and Metzger, Reference De Souza and Metzger1991). Leptin is implicated in both scenarios. Obesity may also adversely affect male fertility and while it is not clear if this involves leptin, Rago et al. (Reference Rago, Aquila, Guido and Carpino2009) recently reported leptin and its receptor in the testis and epididymis of boars.

By all measures (days to first heat, days to first service, services/conception and calving interval) dairy cow fertility has fallen for over 30 years in the United States and United Kingdom, threatening the sustainability and economic viability of herds (Esslemont et al., Reference Esslemont, Kossaibati and Allcock2001). EB is invariably negative in high genetic merit (HGM) dairy cows early PP and sometimes strongly so, well into lactation. Doepel et al. (Reference Doepel, Lapierre and Kennelly2002) and Ferris et al. (Reference Ferris, McCoy, Lennox, Catney and Gordon2002) identified an EB nadir within the first 2-week PP in HGM cows but a state of NEB persisted for several months. Cows with more severe NEB, and leaner primiparous cows, show longer delays to first ovulation (Butler, Reference Butler2005) and follicle growth, oocyte quality and embryo survival are all vulnerable to NEB. Low blood leptin, insulin and IGF-1 may be responsible for the adverse effects (Diskin, Reference Diskin2008). Reist et al. (Reference Reist, Erdin, von Euw, Tschuemperlin, Leuenberger, Chilliard, Hammon, Morel, Philipona, Zbinden, Kuenzi and Blum2002) found leptin only weakly associated with EB (r = −0.027) up to week 10 PP in HGM Holstein cows whereas Wylie et al. (Reference Wylie, Woods, Carson and McCoy2008) found it correlated strongly with mean EB (r = −0.632) up to 3 months of lactation in primiparous HGM cows. Liefers et al. (Reference Liefers, Veerkamp, Te Pas, Delavaud, Chilliard and Van Der Lende2003) saw no relationship between leptin and first PP luteal activity in HGM cows, but acknowledged a possible association between leptin and oestrus expression.

Mann et al. (Reference Mann, Mann, Blache and Webb2005) examined links between leptin and reproductive problems in dairy cows (delayed luteal activity, DLA; cessation of luteal activity, CLA; and prolonged luteal activity, PLA). Problem cows (as identified by milk progesterone) accounted for ∼40% of all cows (DLA, 10.8%; CLA, 18.9%; PLA, 9.5%). Cows with cycle abnormalities had a longer interval to first AI and fewer had conceived by 100-day PP (34.5% v. 66.7% for normal cows). In early lactation, DLA was associated with greater milk yield (MY) and greater loss of weight and condition (implicating NEB in the delayed return of cyclicity) but leptin levels were unrelated to the onset of luteal activity. Between week 6 and week 14 PP, the incidence of abnormal cycles was associated with higher MY, lower leptin and a lower rate of increase in weight and body condition. Konigsson et al. (Reference Konigsson, Savoini, Govoni, Invernizzi, Prandi, Kindahl and Veronesi2008) monitored leptin weekly from 1-week pre partum to 7 weeks PP in primiparous Swedish Red and White cows and found no relationship between leptin in cows that did, and those that did not, resume ovarian activity by the end of week 7 PP. However, this study involved only 12 cows.

Body condition and milk yield are related in sows, as in cows (Butler, Reference Butler2005), and leptin correlates with backfat thickness (r = 0.342) in sows at farrowing. Cyclic activity resumed earlier in sows with higher leptin on day 5 PP (Summer et al., Reference Summer, Saleri, Malacarne, Bussolati, Beretti, Sabbioni and Superchi2009) so that leptin may link metabolic status and fertility in sows also. Xu et al. (Reference Xu, Wu, Guo, Zheng and Zhang2009) fed Landrace × Yorkshire gilts at 0.6, 1.2 or 2 M after mating and found more viable embryos and the highest 25-day embryo survival rate (80%) in gilts fed at 1.2 M. Plasma leptin levels reflected the feed intake. Leptin given to rabbit does for 1-week before inducing ovulation did not affect offspring number or weight but did increase the proportion of pregnant females and numbers of live offspring (Sirotkin et al., Reference Sirotkin, Rafay and Kotwica2009).

Leptin response to parasite infection in farm animals

Ruminants suffer recurrent gut parasite infections that cause under-performance (Kulcsár et al., Reference Kulcsár, Jánosi, Lehtolainen, Kátai, Delavaud, Balogh, Chilliard, Pyörälä, Rudas and Huszenicza2005) due to competition for nutrients between the immune system and growth (Colditz, Reference Colditz2002) and anorexia is a common feature of nematode infection. Well-nourished animals are better able to resist or eliminate infection, perhaps via leptin’s effects on T-cells and proinflammatory cytokines IL-6 and TNF-α (Lord et al., Reference Lord, Matarese, Howard, Baker, Bloom and Lechler1998). Leptin levels typically rise in infection (Faggioni et al., Reference Faggioni, Feingold and Grunfeld2001) and may mediate the parasite-induced anorexia seen in sheep. Amarante et al. (Reference Amarante, Bricarello, Rocha and Gennari2004) found faster-growing Suffolk lambs more susceptible than slower-growing breeds, while they also suffer more intense and longer anorexia (Sandberg et al., Reference Sandberg, Emmans and Kyriazakis2006). Liu et al. (Reference Liu, Adams, Briegel, Smith and Martin2007a) saw a non-significant rise in leptin in restricted Merinos infected with T. colubriformis and Teladorsagia circumcincta. Zaralis et al. (Reference Zaralis, Tolkamp, Houdijk, Wylie and Kyriazakis2008) infected Scottish Blackface and Suffolk × Greyface lambs with T. circumcincta for 12 weeks and noted a 13% reduction in intake in Suffolk × Greyface but no loss of intake in Scottish Blackface (a breed relatively resistant to parasite infection; Coop and Holmes, Reference Coop and Holmes1996). Zaralis et al. (Reference Zaralis, Tolkamp, Houdijk, Wylie and Kyriazakis2008) also found higher leptin in infected lambs than in non-infected lambs restricted to similar intake (but not in those fed ad libitum). Greer et al. (Reference Greer, Boisclair, Stankiewicz, McAnulty, Jay and Sykes2009) noted a 20% fall in intake but no change in leptin level in lambs infected by T. colubriformis, while calves infected with Ostertagia ostertagi (10 000 larvae/day) had lower leptin but with no change in intake or weight gain (Forbes et al., Reference Forbes, Warren, Upjohn, Jackson, Jones, Charlier and Fox2009). Differences in dietary energy and protein may explain some of the anomalies in these studies as protein counteracts some effects of gut parasites in sheep (Forbes et al., Reference Forbes, Warren, Upjohn, Jackson, Jones, Charlier and Fox2009) but Zaralis et al. (Reference Zaralis, Tolkamp, Houdijk, Wylie and Kyriazakis2009) showed that supplementary protein did not affect leptin levels or the degree of anorexia in ewes infected with T. circumcinta.

Depressed immune function in the periparturient cow makes it vulnerable to infection (Elsasser et al., Reference Elsasser, Ingvartsen, Kahl and Capuco2006). The extent of the depression is important and the early PP fall in leptin might play a part (Ingvartsen and Boisclair, Reference Ingvartsen and Boisclair2001). Loiselle et al. (Reference Loiselle, Ster, Talbot, Zhao, Wagner, Boisclair and Lacasse2009) milked Holstein cows once or twice daily in week 1 PP and assessed leucocytic cytokine ouput: as expected, leptin levels fell after calving, but less so in cows milked only once daily. Higher INF-γ in unmilked cows was consistent with lower immunosuppression, but the study did not prove a causal link to leptin.

The significance of leptin gene SNPs in livestock

SNPs in exons and introns of bovine and porcine LEP (and the bovine promoter) associate with intake, liveweight (LW) gain, carcase quality and reproductive performance in pigs and beef cattle and with milk yield in cows (Van der Lende et al., Reference Van der Lende, Te Pas, Veerkamp and Liefers2005). Most associate also with blood leptin levels but the strength of these associations may be different under different physiological states (e.g. growth, gestation and lactation).

Beef cattle

Fitzsimmons et al. (Reference Fitzsimmons, Schmutz, Bergen and McKinnon1998) investigated a microsatellite (BM1500) ∼3.6 kb downstream of the LEP stop codon in purebred beef bulls (Angus, Charolais, Hereford and Simmental) and showed links between polymorphisms in BM1500 and carcase fat. Buchanan et al. (Reference Buchanan, Fitzsimmons, van Kessel, Thue, Inkelman-Sim and Schmutz2002 and Reference Buchanan, van Kessel, Waldner, Christensen, Laarveld and Schmultz2003) found greater carcase fat and leptin mRNA expression in cattle T-homozygous for C305T (LEP exon 2) that encodes an Arg-Cys change at aa-4 in mature leptin. This suggested higher feed intake by TT genotypes. In Angus, Charolais and Hereford, TT genotypes had more backfat at slaughter, attained slaughter quality at lighter weight and 16 days earlier than CC genotypes and blood leptin was correlated (r = 0.45) with backfat depth (Buchanan et al., Reference Buchanan, van Kessel, Boisclair, Block and McKinnon2007). Kononoff et al. (Reference Kononoff, Deobald, Stewart, Laycock and Marquess2005) reported comparable data for >1500 crossbred heifers and steers with ∼7% more TT than CC or CT genotypes having carcases with high marbling (AAA grade or higher) and 12% to 15% fewer TT than CT or CC genotypes with maximum leanness.

Other LEP SNP in beef cattle include A1457G and C963T (Liefers et al., Reference Liefers, Veerkamp, Te Pas, Chilliard and Van Der Lende2005), C207T and C528T (Nkrumah et al., Reference Nkrumah, Li, Yu, Hansen, Keisler and Moore2005) and A252T (Lagonigro et al., Reference Lagonigro, Wiener, Pilla, Woolliams and Williams2003) while LEP exon-3 SNP (A59V) associated with weight at 210 days and mean gain to 210 days in Limousins, suggesting its usefulness in breeding for heavier Limousin carcases (Kulig and Kmieć, Reference Kulig and Kmieć2009). Schenkel et al. (Reference Schenkel, Miller, Ye, Moore, Nkrumah, Li, Yu, Mandell, Wilton and Williams2005) explored links between four SNP (UASMS1 (C207T), UASMS2 (C528T), E2JW (A252T) and E2FB (C305T)) and fat and lean yield and meat quality characteristics (im fat; L. dorsi and M. semitendinosus tenderness) in >1100 crossbred bulls, heifers and steers (Angus, Simmental, Charolais and Limousin). E2JW and E2FB associated with ‘fat and lean yield’ and interacted in effects on L. dorsi tenderness while UASMS1 promoter polymorphism associated only with fat yield. Haplotypes may be superior predictors of carcase characteristics in cattle (Stone et al., Reference Stone, Casas, Smith, Keele, Harhay, Bennett, Koohmaraie, Wheeler, Shackelford and Snelling2005) and TCAC, CCAT and TTAC similarly affected all traits while CCTT associated mostly with ‘fat and lean yield’ and TTTT with L. dorsi tenderness (Schenkel et al., Reference Schenkel, Miller, Ye, Moore, Nkrumah, Li, Yu, Mandell, Wilton and Williams2005).

Gill et al. (Reference Gill, Bishop, McCorquodale, Williams and Wiener2009) studied eight SNP (three in LEP; UASMS1, UASMS2 and exon 2FB) and their association with meat quality in Aberdeen Angus cattle. These SNPs are already incorporated to varying degrees in commercial genotyping tests (Igenity^© and Genestar^©) for tenderness, fatness and carcase composition in beef cattle and milk yield and quality in dairy cows. In the study, meat from cattle TT-homozygous for UASMS2 gained higher taste panel scores and this test is one element of the Igenity OptiGRID^© test. Exonic LEP SNPs also associated with carcase parameters and meat quality in Korean Hanwoo assessed either visually (Shin and Chung, Reference Shin and Chung2007) or by ultrasound (Kong et al., Reference Kong, Oh, Lee, Hong, Song, Lee, Kim, Yoo, Lee and Jeon2006). In the former study, SNP C1180T associated with backfat and marbling score, with CC genotypes having greater fat depth than TT and higher marbling score than CT or TT. Marbling has reduced in many cattle breeds in recent decades (Dunshea et al., Reference Dunshea, D’Souza, Pethick, Harper and Warner2005). Current breeding policy seeks to increase marbling without increasing s.c. fat. However, Pannier et al. (Reference Pannier, Sweeney, Hamill, Ipek, Stapleton and Mullen2009) investigated four LEP SNP in nine purebred cattle types and found no significant association between any of them (or 16 possible haplotypes) and i.m. fat. QTL for marbling and s.c. fat in beef cattle reside on different genes and may allow selection for higher marbling and lower s.c. fat. Importantly, also, QTL linked to marbling show breed specificity (Jiang et al., Reference Jiang, Pappu and Rothschild2007) being on chromosomes BTA2, BTA3, BTA16, BTA17, BTA23, BTA27, and BTA29 in US beef breeds but on BTA4, BTA6, BTA7 and BTA13 in Japanese Wagyu and Korean Hanwoo.

Dairy cows

Buchanan et al. (Reference Buchanan, van Kessel, Waldner, Christensen, Laarveld and Schmultz2003) found LEP C305T in six dairy breeds. Cows with one or more T alleles had higher MY with TT cows giving 1.5 kg/day more milk than CC cows across lactation (2.44 kg/day to day 100). A similar effect in beef cows should favour heavier or earlier weaned calves (De Vuyst et al., Reference De Vuyst, Bauer, Cheng, Mitchell and Larson2008). Buchanan et al. (Reference Buchanan, van Kessel, Waldner, Christensen, Laarveld and Schmultz2003) also noted higher milk protein yield in TT than CC cows over all of lactation. Liefers et al. (Reference Liefers, Te Pas, Veerkamp and Van Der Lende2002a) genotyped >600 Holstein heifers for two restriction fragment length polymorphisms (RFLP) and the BM1500 microsatellite and associated them with intake, EB, LW and MY to 15-week PP, and with time to commencement of luteal activity. RFLP1-AB heifers gave 1.32 kg/day more milk and ate 0.73 kg/day over RFLP1-AA heifers but time to commencement of luteal activity was unaffected. Selection for RFLP1-AB might allow higher MY without lowering fertility. Banos et al. (Reference Banos, Woolliams, Woodward, Forbes and Coffey2008) associated LEP and LEPR SNP in Holsteins with intake, MY, feed efficiency, LW and body condition and identified a haplotype (C207T, C528T, A1457G, C963T, A252T and C305T) linked to a MY bias of 3.13 kg/day and a feed intake increment of 4.64 kg/day. Importantly, cows with the haplotype tended to retain more body energy in early lactation, suggesting greater likelihood of superior fertility due to lower loss of fat and condition. They concluded that dairy traits are affected in small ways by many genes. Clearly, genotyping for lactational performance need not be confined to females. Madeja et al. (Reference Madeja, Adamowicz, Chmurzynska, Jankowski, Melonek, Switonski and Strabel2009a) explored the LEP HphI RFLP in Polish Black and White bulls and found TT bulls had almost twice the estimated breeding value for MY and milk fat and protein.

Five other LEP SNPs were listed by Van der Lende et al. (Reference Van der Lende, Te Pas, Veerkamp and Liefers2005) and associations shown for Tyr7Phe, LEPSau3AI and Arg25Cys with intake, MY, carcase fat and reproductive success. Liefers et al. (Reference Liefers, Veerkamp, Te Pas, Delavaud, Chilliard and Van Der Lende2002b) showed traits that were linked with serum leptin level in late gestation but not during lactation, whereas Liefers (Reference Liefers2004) screened 14 of 18 LEP promoter SNPs for links with production and reproduction and found G(-282)T associated with serum leptin in late pregnancy and early lactation while C(-201)T associated with leptin levels only in early lactation. Three others associated with production or reproduction: C(-963)T with intake, EB and calving to 1st oestrus interval; A(-1457)G with days to commencement of luteal activity and early-PP weight loss and C(-578)G with percent milk protein.

Sheep

Boucher et al. (Reference Boucher, Palin, Castonguay, Gariepy and Pothier2006) reported three LEP SNPs in Dorset and Suffolk lambs. A103G associated with thickness and shear force of M. longissimus in Suffolks and metabolic activity in M. longissimus and s.c. fat in Dorsets. Zhou et al. (Reference Zhou, Hickford and Gong2009) found three SNPs in exon 3 that result in aa changes and could affect leptin structure and function.

Pigs and poultry

Jiang and Gibson (Reference Jiang and Gibson1999) identified four SNP (A2845T, T3469C, G2728A and T3996C) in porcine LEP. All were poorly associated with backfat thickness. These same SNP were absent or of low frequency in Durocs, Landrace and Yorkshire breeds (Kennes et al., Reference Kennes, Murphy, Pothier and Palin2001) while A2845T and T3469C associated with intake and growth rate in Landrace only. In pig LEP, C867T, A2845T and T3469C associated with production and reproduction traits (Van der Lende et al., Reference Van der Lende, Te Pas, Veerkamp and Liefers2005) but Amills et al. (Reference Amills, Villalba, Tor, Mercade, Gallardo, Cabrera, Jimenez, Noguera, Sanchez and Estany2008) genotyped Landrace for exon-3 C3469T and found no associations between TT or TC and plasma leptin level, growth rate or carcase fatness. Associations of C3469T with i.m. fat and body composition of Durocs (Villalba et al., Reference Villalba, Tor, Vidal, Bosch, Reixach, Amills, Sanchez and Estany2009), varied with age (160 to 225 days) and may explain the variable data for this SNP. Promoter region SNP do not associate with leptin expression or fatness in Durocs (Stachowiak et al., Reference Stachowiak, Mackowski, Madeja, Szydlowski, Buszka, Kaczmarek, Rubis, Mackowiak, Nowak and Switonski2007) but Peixoto et al. (Reference Peixoto J de, Guimarães, Lopes, Soares, Pires, Barbosa, Torres R de and e Silva M de2006) found some carcase characteristics associated with T2411C and T3266G, making them potential markers of pig carcase composition. Out of >800 QTL in pigs, 500 were shown to be associated with fat deposition, for example, carcase fat, abdominal fat, backfat thickness, i.m. fat and intermuscular fat (Rothschild et al., Reference Rothschild, Hu and Jiang2007). Marbling and i.m. fat are correlated (r = 0.57) but are linked to different QTL (Huff-Lonergan et al., Reference Huff-Lonergan, Baas, Malek, Dekkers, Prusa and Rothschild2002) with marbling QTL on SSC1, 8, 10, 12, 14 and 17 and those for i.m. fat on SSC4 and SSC7 and the X chromosome. Only SSC6 has QTL for both the features.

Despite the sequencing of the entire chicken genome (International Chicken Genome Sequencing Consortium, 2004) and the identification of ∼2.8 m SNP (International Chicken Polymorphism Map Consortium, 2004) there are few reports, yet, of polymorphisms in ch-LEP.

Leptin receptor gene SNPs

The 20-exon leptin receptor (LEPR) gene is another candidate gene for production traits in livestock. A missense mutation at position 115 of bovine LEPR exon 20 causes a threonine to methionine change in the leptin receptor in Holstein-Friesian cows (Liefers, Reference Liefers2004) giving allele frequencies of 0.93 (C) and 0.07 (T) with no TT. Blood leptin levels were influenced by this LEPR SNP in late pregnancy but not in lactation with CC cows having higher levels than CT. It associated also with milk fat and protein percentage (but not with yields of milk, milk fat or milk protein) in Jerseys (Komisarek and Dorynek, Reference Komisarek and Dorynek2006). Almeida et al. (Reference Almeida, Santos, Passos, Corbellini, Lopes, Kirst, Terra, Neves, Goncalves, Moraes and Weimer2008) investigated it in Angus, Brangus and Charolais cows and found no associations with reproductive characteristics. Schenkel et al. (Reference Schenkel, Miller, Ye, Moore, Li, Yu, Lobo, Mandell and Wilton2006) reported only one LEPR SNP associated with s.c. fat mass, fat yield and fat grade in beef cattle but Guo et al. (Reference Guo, Chen, Lan, Zhang, Pan, Zhang, Zhang and Zhao2008) genotyped LEPR exon 4 SNP in five Chinese breeds and found strong influences on height, weight, length and weight gain at 6 and 12 months.

Vincent et al. (Reference Vincent, Wang and Rothschild1997) first reported a polymorphism in porcine LEPR, one of several genes linked to fat deposition in pigs (Ovilo et al., Reference Ovilo, Fernandez, Noguera, Barragan, Leton, Rodriguez, Mercade, Alves, Folch, Varona and Toro2005). Amills et al. (Reference Amills, Villalba, Tor, Mercade, Gallardo, Cabrera, Jimenez, Noguera, Sanchez and Estany2008) found no association between an RFLP in LEPR and growth rate or fatness in >300 Landrace pigs but Sun et al. (Reference Sun, Wang, Jiang and Zhang2009) associated SNP C155T in LEPR exon 2 with first litter size and percentage of live piglets in Large White sows. Birth weight, weaning weight and neonatal weight gain were unaffected. Muñoz et al. (Reference Muñoz, Alves, Fernández, Óvilo, Barragán, Estellé, Quintanilla, Folch, Silió, Rodríguez and Fernández2009b) associated an SSC6 QTL with growth and backfat thickness in Iberian × Meishan and Iberian × Landrace pigs. A QTL linked to liveweight also was found in each crossbreed while an exon 14 SNP also associated with backfat thickness in each. In chickens, one of two recently found SNP in exon 9 of ch-LEPR has been linked to abdominal fat in broilers (Wang et al., Reference Wang, Li, Zhang, Gu, Li and Wang2006).

Genomic identity is established at conception and is a lifelong constant. Genomic breeding value provides a neonatal assessment of production potential (Hayes et al., Reference Hayes, Bowman, Chamberlain and Goddard2009) so that bulls could be used as early as 2-years of age, saving hugely on progeny-testing costs (Schaeffer, Reference Schaeffer2006). Data volumes in such work are staggering with 38 259 out of a total of 56 947 SNP genotyped in ∼800 Holstein-Friesian bulls in one Australian programme. In similar US, New Zealand and Dutch work about 38 000, 44 000 and 46 000 SNP were genotyped in ∼3500, 4500 and 1500 progeny-tested bulls, respectively (Hayes et al., Reference Hayes, Bowman, Chamberlain and Goddard2009).

Leptin SNPs and immune responsiveness

Associations between leptin SNP and immune responses might help predict susceptibility to disease. Asiamah et al. (Reference Asiamah, Betchel, Waldner and Buchanan2009) used antibody response to rabies to explore links between an SNP associated with carcase quality in beef cattle and milk composition in cows. Genotypes did not differ in antibody response so that using this SNP to select for production should not dramatically impair immune function. Chebel et al. (Reference Chebel, Susca and Santos2008) looked at associations of exon-2 R4C with common PP problems in Holstein cows (e.g. retained placenta, displaced abomasum and mastitis). Genotype had little effect on the incidence of any single problem except displaced abomasum for which TT cows had a higher incidence than CT cows (which had the lowest risk of suffering any of the conditions).

Leptin and leptin receptor SNPs and reproduction

Kuehn et al. (Reference Kuehn, Nonneman, Klindt and Wise2009) investigated associations of a LEP SNP and two LEPR SNPs with age, weight, backfat and leptin level at puberty in three gilt lines. Age and leptin level were correlated (r = −0.63) and one of the LEPR SNPs was associated with blood leptin levels, though not necessarily causatively.

Endogenous leptin as a marker of body composition

Blood leptin level broadly reflects fat level in growing animals but the relationship is not robust (Altmann and von Borell, Reference Altmann and von Borell2007). Age and nutrition effects (especially fasting) make a one-size-fits-all approach untenable but it may still find use in predicting carcase fat within large groups of genetically similar animals near or at slaughter.

Endogenous leptin as a marker of reproductive readiness in females

In livestock farming, EB is probably of most practical significance in the peripartum dairy cow. Reproductive success (conception to weaning) is more assured in fatter animals (Friggens, Reference Friggens2003) so that the association of leptin with body condition and EB in ruminants (Chilliard et al., Reference Chilliard, Delavaud and Bonnet2005) suggests leptin levels might mark the readiness of recently calved cows to resume breeding. Milk producers aim for a lactation of ∼10 months and a dry period of ∼6 or 8 weeks (during which a cow repletes fat reserves for the next lactation) giving an idealised calving interval of close to 1 year. Higher genetically driven MY has been accompanied by longer and deeper PP NEB and a lengthening of the calving interval, as either the depth or duration of the NEB or the number of days to the EB nadir affect the time to resumption of oestrus activity (Beam and Butler, Reference Beam and Butler1999). Leptin is a potential marker of EB and the EB nadir: Block et al. (Reference Block, Butler, Ehrhardt, Bell, Van Amburgh and Boisclair2001) milked cows 3 × daily or left them unmilked up to week 8 PP and found that milked cows experienced severe NEB (−70.3 MJ/day average, 1 to 4 weeks) while unmilked cows remained in positive EB (50.6 MJ/day average) with mean plasma leptin of 2.9 and 5.6 ng/ml, respectively. However, Kadokawa et al. (Reference Kadokawa, Blache, Yamada and Martin2000) found ‘days from parturition to leptin nadir’ correlated strongly (r = 0.83) with ‘days to first PP ovulation’ while Liefers et al. (Reference Liefers, Veerkamp, Te Pas, Delavaud, Chilliard and Van Der Lende2003) saw no clear leptin nadir in >300 primiparous Holsteins to 80-day PP. Such discrepancies remain unresolved, but using PP leptin levels to assess the readiness of dairy cows for renewed mating remains a credible prospect if milk leptin (assayed by an in-line biosensor) can be reliably related to blood concentrations.

Endogenous leptin as a marker of meat eating quality

Wagyu and Korean Hanwoo cattle produce meat of exceptional eating quality. Leptin concentrations are inversely related to i.m. fat in these breeds, and in crossbreeds in which they are dominant, and remain a potential marker of marbling. The fatty acids in beef fat triglycerides influence eating quality and oleic acid and PUFA contents are higher in breeds of higher eating quality (see review by Hausman et al. (Reference Hausman, Dodson, Ajuwon, Azain, Barnes, Guan, Jiang, Poulos, Sainz, Smith, Spurlock, Novakofski, Fernyhough and Bergen2009)) but no association has yet been found between leptin and fatty acid composition in i.m. fat, or in any other body fat depot.

Endogenous leptin as a marker of immune capability

Valderrabano et al. (Reference Valderrabano, Gomez-Rincon and Uriarte2006) infected 90-day pregnant ewes weekly, to the end of pregnancy, with highly pathogenic Haemonchus contortus and found worm burdens lower in fatter ewes. The stronger immune response may have been linked to higher leptin concentrations in the fatter ewes – a potentially valuable observation in the context of increasing anthelminthic resistance to chemical controls and general unease about chemical use.

Exogenous leptin to manipulate performance

Leptin-induced fat loss, while retaining lean mass, has clear clinical applications in man (e.g. pre-surgical weight loss) but is not an option in farm animals on any grounds (welfare, practicality or cost). However, there are other ways in which leptin might be used to influence production quality and production efficiency:

Exogenous leptin to advance the onset of puberty

Studies in man, rodents and farm species all suggest leptin is permissive rather than causal to puberty onset with a threshold level necessary only to allow puberty to proceed. Advancing the age at which puberty occurs should increase lifetime production and reproductive output but Maciel et al. (Reference Maciel, Zieba, Amstalden, Keisler, Neves and Williams2004) increased leptin (7 ×) in prepubertal heifers without inducing puberty. However, Paczoska-Eliasiewicz et al. (Reference Paczoska-Eliasiewicz, Proszkowiec-Weglarz, Proudman, Jacek, Mika, Sechman, Rzasa and Gertler2006) injected re-ch.leptin daily into 11-week pullets and advanced the age of first lay by day 5, showing potential for increasing lifetime productivity from layers.

Exogenous leptin to assure physiological readiness for breeding

Delays in recommencing reproductive activity in PP dairy cows is seen as a reaction to an event (pregnancy) that the cow has judged as unsustainable in energy terms. Increasing feed availability early PP might be expected to alleviate the energy deficit, but HGM cows direct more of their EI to milk production (Yan et al., Reference Yan, Mayne, Keady and Agnew2006) so that, paradoxically, NEB may even be exacerbated. A delayed return to cyclicity in dairy cows has been linked to low insulin levels but occurs during a period of low leptin also. Garnsworthy et al. (Reference Garnsworthy, Fouladi-Nashta, Mann, Sinclair and Webb2009) improved oocyte quality and pregnancy rate in cows by feeding for higher insulin until cycling resumed and then changing the diet to promote lower insulin at, and after, insemination. They found no change in plasma leptin level, but exogenous leptin might allow hypothalamogonadal activity ahead of a substantive fall in NEB. The model is the restoration of reproductive axis activity in ob mice by leptin (Chehab et al., Reference Chehab, Lim and Lu1996) and the strategy is to re-establish blood leptin to a level that convinces integrative centres in the brain that fat stores are sufficient to support a pregnancy.

The possibilities for exogenous leptin use (above) are predicated on increasing leptin concentrations in blood but immunising against LBP might have the same result. Conversely, immunising against leptin might allow increased intake and production. Wuethrich et al. (Reference Wuethrich, Hancock, Long, Manetta, Roth and Anderson1998) immunised 170 kg gilts against leptin using aa 1 to 20 of mouse leptin but, despite high-titre antibody, they did not change feed intake whereas Shi et al. (Reference Shi, Shao, Chen, Yu, Bi, Liang, Williams and Taouis2006) immunised hens and pullets against leptin and increased intake and abdominal fat by 66% in pullets and fat by ∼50% in hens, while also reducing laying performance.

Exploiting leptin in farm species may require cheaper and more robust leptin analogs and novel delivery systems. Grasso et al. (Reference Grasso, Leinung, Ingher and Lee1997) localised leptin’s effects on appetite to aa106 to aa140, but Nonaka et al. (Reference Nonaka, Hashizume and Yamashita2006) could not replicate LH induction in pig pituitary cells using aa116 to aa130. With regard to delivery, Chen et al. (Reference Chen, Koyama, Yuan, Lee, Zhou, O’Doherty, Newgard and Unger1996) used recombinant adenovirus to transfer the leptin gene into rats, increasing leptin expression and causing fat loss and this may be a viable way to promote leptin expression in pigs also (Barb et al., Reference Barb, Hausman and Houseknecht2001b).

Applying LEP or LEPR gene polymorphisms to improving performance

The use of DNA-based marker assisted selection has been slower to develop in animal agriculture than in crop agriculture due to the many gene loci involved in complex traits like milk production (Hayes et al., Reference Hayes, Bowman, Chamberlain and Goddard2009) making extensive genotyping necessary. This has been made easier by the sequencing of the bovine genome (Bovine Genome Sequencing and Analysis Consortium, 2009) and chicken genome (International Chicken Genome Sequencing Consortium, 2004) and by high genotyping capacity and falling genotyping costs. The leptin gene is a candidate gene for fat deposition and meat quality. With marbling a major contributor to meat quality, the challenge is to increase i.m. fat while avoiding increases in s.c. fat and other fats. LEP gene SNPs (and other gene SNPs) may enable early detection of a genotype that favours i.m. fat deposition, allowing nutrition to be customised to produce more marbling (Hocquette et al., Reference Hocquette, Lehnert, Barendse, Cassar-Malek and Picard2007 and Reference Hocquette, Gondret, Baeza, Medale, Jurie and Pethick2010). Commercial SNP tests can already identify cattle with higher potential for leanness or milk yield. And while associations of LEP SNP with milk yield and milk quality have proven value, the recent association of C305T with weaning weight and maternal longevity (Mitchell et al., Reference Mitchell, DeVuyst, Bauer and Larson2009) might also enhance profitable calf rearing. Improving cow health while sustaining milk production is also desirable and any links between LEP SNP and disease susceptibility will be valuable. These qualities are related through interaction of a cow’s genotype for MY and its energy status, i.e. cows with adequate fat will more easily satisfy the energy requirement for genetically driven yield and suffer less severe NEB than those of similar genetic potential, but lesser fat, with a likelihood of earlier ovulation and a lower risk of metabolic or infectious disease.

Early bovine leptin SNP studies mostly targetted Bos taurus, but associations with production are now evident in Bos indicus breeds. Corva et al. (Reference Corva, Fernández Macedo, Soria, Papaleo Mazzucco, Motter, Villarreal, Schor, Mezzadra, Melucci and Miquel2009) investigated two leptin gene SNPs (one in exon 2; one in the promoter) in Brangus (5/8 Angus, 3/8 Brahman) steers and found associations with backfat thickness, rib-eye muscle area and carcase yield. Lower marbling in B. indicus is important, but Fortes et al. (Reference Fortes, Curi, Chardulo, Silveira, Assumpção, Visintin and de Oliveira2009) failed to find associations between leptin SNPs and backfat, rib-eye area or marbling score in purebreds or B. indicus × B. taurus crossbreeds, and there was no association with shear force (tenderness) which is lower in B. indicus. The validity of applying B. taurus gene markers to B. indicus is not yet clear and new markers may need to be developed for B. indicus.

Feed intake, conversion efficiency and carcase quality are equally important in pig production. Associations of LEP and LEPR SNP with age and weight at puberty in gilts (Kuehn et al., Reference Kuehn, Nonneman, Klindt and Wise2009) suggests possible QTL to select for earlier puberty in pigs but many breeds are used worldwide with different strengths of association between LEP SNP and production and reproduction traits. D’Andrea et al. (Reference D’Andrea, Pilla, Giuffra, Waddington and Archibald2008) identified 18 LEP haplotypes with different distributions between fat and lean breeds suggesting unwitting selection for LEP SNP over time. This may explain anomalies in earlier pig studies, which considered only single SNP. There are opportunities also to apply LEP and LEPR SNP to improving poultry production, once the current uncertainties about ch-LEP are resolved.

Few associations have yet been made between LEPR SNP and production traits in Western dairy and beef cattle but recently established links with body measures in Chinese cattle may speed productivity increases in these demographically important breeds. Associations of a LEPR exon-2 SNP (C155T) in Large Whites with first litter size and percentage of liveborn might help selection for reproductive output in pigs. The balance between hunger and satiety has changed in modern broilers (Richards, Reference Richards2003) with selection for rapid growth accompanied by resistance to anorexigenic signals from the periphery (Cassy et al., Reference Cassy, Picard, Crochet, Derouet, Keisler and Taouis2004), giving hyperphagic birds that readily accumulate fat (Bokkers and Koene, Reference Bokkers and Koene2003). Reducing fat without risking the established genetic gains in growth is challenging, but necessary, and associations of fat with recently found ch-LEPR SNP might help in broiler breeder selection.

Neonatal livestock performance

Leptin’s involvement in immune responses makes it a potential candidate for monitoring and/or intervention to ensure satisfactory progress in neonates and peripartum females in which disease is a serious welfare concern. In pigs, decades of intensive genetic selection has brought larger litters but more very low birth weight pigs (<1 kg) and higher within-litter birth weight variation (Morise et al., Reference Morise, Louveau and Le Huërou-Luron2008). Very low birth weight pigs (runts) account for 10% to 15% of littermates with mortality statistics typically between 9% and 12% of all neonates (Ramsay et al., Reference Ramsay, Stoll and Caperna2010), but as high as 50%. Leptin and its receptor mRNAs are detectable in s.c. fat in neonatal pigs (Chen and Heiman, Reference Chen and Heiman2000) suggesting that piglet survival and performance might be influenced by leptin and it may be significant that Litten et al. (Reference Litten, Mostyn, Laws, Corson, Symonds and Clark2008) gave leptin i.v. to 6-day old pigs and altered their ability to thermoregulate. Attig et al. (Reference Attig, Djiane, Gertler, Rampin, Larcher, Boukthir, Anton, Madec, Gourdou and Abdennebi-Najar2008) showed that runt piglets have altered leptin receptor distribution in hypothalamic centres involved in metabolic regulation. Leptin supplementation partially corrected the runt phenotype (normalising food intake and post-natal growth and reducing the increased adiposity) and normalised the development of organs involved in metabolic regulation. Ramsay et al. (Reference Ramsay, Stoll and Caperna2010) reported differences between early neonatal runt piglets and control piglets in respect of leptin mRNA levels in s.c. adipose tissue on day 1 (runts <controls) and in perirenal adipose tissue on day 7 (runts > controls) but levels of many other adipokine mRNAs were also differentially affected. Lambs born to ewes restricted in early or mid-pregnancy developed higher leptin levels and males deposited more fat as adults (Muñoz et al., Reference Muñoz, Carson, McCoy, Dawson, Wylie and Gordon2009a). Similar data in rats (Vickers et al., Reference Vickers, Gluckman, Coveny, Hofman, Cutfield, Gertler, Breier and Harris2005) point to ‘developmental programming’ or ‘foetal programming’ with lifetime health and performance implications.

Welfare implications

The public often perceive thin dairy cows as undernourished and a welfare concern. Paradoxically, overfat cows can be more problematic and succumb more readily to periparturient disease (Treacher et al., Reference Treacher, Reid and Roberts1986). The LEP allele associated with higher MY in Holsteins has been linked to a small increase in metritis (Oikonomou et al., Reference Oikonomou, Angelopoulou, Arsenos, Zygoyiannis and Banos2009) but may offer a tool to improve the management of periparturient cows, ewes and sows and reduce the incidence of metabolic and infectious PP diseases. The possibility that low early-PP leptin concentrations might increase disease susceptibility needs investigation and could lead to novel therapeutic or prophylactic applications for leptin.

Environmental impact (carbon footprint, etc.)

Energy efficiency is fundamental to profitable animal production and is central to all initiatives to lower animal contributions to climate change. Feed evaluation and EE have been research focusses for most of the past century (Johnson et al., Reference Johnson, Ferrell and Jenkins2003) while variation in energetic efficiency of individuals has received rather less attention, but may be a lucrative route to future improvements. RFI (the difference between the actual and expected feed intake for a given level of performance) is one useful measure of efficiency. Leptin levels were positively associated with RFI in beef steers (Nkrumah et al., Reference Nkrumah, Keisler, Crews, Basarab, Wang, Li, Price, Okine and Moore2007) but not in beef heifers (Kelly et al., Reference Kelly, McGee, Crews, Fahey, Wylie and Kenny2010). Associations of leptin with RFI could be extremely valuable for genetic improvement of Bos indicus breeds and LEP SNPs in B. indicus breeds might enable selection for more meat and milk output from indigenous cattle in regions in which man’s consumption of animal protein is growing, but from a low base.