Physiology and genetics of leptin in periparturient dairy cows

In dairy cattle, the increase in milk yield has been accompanied by a decrease in fertility and a more negative energy balance. As the hormone leptin is involved in regulation of nutritional status and reproductive function (Chapter 2) this is an interesting protein to investigate during the periparturient period in dairy cattle when many changes take place both in energy metabolism and reproductive physiology. The objectives of this study weretoget insight into the function of leptin during the periparturient period and to perform an association study between polymorphisms in the bovine leptin and leptin receptor gene and fertility and production traits.The leptin gene including its promoter region and parts of the leptin receptor gene were sequenced to find polymorphisms,and related to differences in fertility and production traits.Leptin levels were described during the periparturient period in dairy cows and also associations with the genotyped polymorphisms and fertility and production traits were calculated.In Chapter 2 a literature overview regarding leptin and its receptor and their role in metabolic processes and fertility was given. This Chapter also includes recent literature, which was published during the investigations that were described in other Chapters of this thesis.Physiology of LeptinIn Chapter 4 leptin levels during the periparturient period in dairy cows were described and these levels were related to differences in production and reproduction traits. Leptin concentrations in the periparturient cow undergo remarkable changes; leptinconcentrations were high during late pregnancy and declined to a nadir at parturition.The reason of elevated plasma leptin during pregnancy in ruminants seems to be the increase in adipose tissue leptin mRNA due to the lack of a negative feedback (leptin resistance state) and the increase in adiposity. We observed an association of live weight during lactation and prepartum leptin concentrations (Chapter 4) but did not investigate the association between prepartum live weight and leptin concentrations.The reason of the decline in leptin levels towards parturition and during lactation is the mobilization of adipose tissue. Leptin concentrations seem to reflect the state of energy balance during lactation; plasma leptin concentrations were lower in cows with a mean negative energy balance during lactation (Chapter 4).Further analysis indicated that a combination of three polymorphisms located at the leptin promoter region explained 14.3% of the variance in prepartum leptin concentrations. The two extreme combinations with the highest and lowest prepartum leptin concentrations could be used to investigate the function of leptin concentrations in pregnant cows.Genetics of LeptinLeptin influences feed intake, energy balance and fertility and therefore the leptin gene and the leptin receptor gene are possible candidate genes to investigate effects on energy balance and fertility in lactating cows. The leptin gene including its promoter region and parts of the leptin receptor gene were sequenced to find polymorphisms, but also to detect putative transcription factor binding sites on the leptin promoter region. An association study was performed with the found polymorphisms. Associations of allgenotyped polymorphisms with fertility and production traits measured in dairy cows during the periparturient period (Chapters 3 and 7) and alsodifferences in leptin levels between genotypes of all genotyped polymorphisms were analyzed (Chapters 5, 6, and 7).An intronic polymorphism (RFLP1) located on intron 2 of the leptin gene explained a significant part of the variation in milk yield, with differences of 0.5 genetic standard deviation between the two extreme genotypes (Chapter 3). However, because this polymorphism is located at an intronic region and because the polymorphisms found in the exons of the leptin gene were not associated with milk yield, we can not exclude that the causative gene is in linkage disequilibrium with this polymorphism. On the promoter region of the leptin gene SNP -1457 was associated with first postpartum luteal activity ( p =0.017) and with weight loss between week 1 and its minimum weight ( p =0.027), where more weight loss occurred together with a later first postpartum luteal activity.Fertility traits are considered to be important to select for and this SNP could be a potential candidate marker to be informative for fertility in dairy cows. SNP -963 was associated with energy balance ( p =0.015) and dry matterintake(p = 0.030), where a higher dry matter intake occurred together with a higher energy balance (Chapter 7).In order to find an optimal genotype combination with a high milk yield, a good energy balance and fertility some new analysis were performed in Chapter 8. Two genotype combinations of three SNPs were defined in this Chapter and a next experiment and calculations of economical values per trait have to validate if one of these genotype combinations would be a possible candidate to be used in selection.The R4C polymorphism, located at exon 2 of the leptin gene, received a lot of attention by several research groups because of its putative effect on the leptin structure, but several studies, including our study, did not provide convincing evidence that the R4C polymorphism influences the structure and function of leptin. Other studies showed an effect on milk yield in dairy cattle and carcass fat content in beef cattle. On the basis of these results a commercially available R4C-test was developed to improve breeding values in both dairy and beef cattle.Polymorphisms in the leptin and leptin receptor gene have shown to be associated with differences in leptin concentration during late pregnancy (Chapters 5 and 6) but, surprisingly, not during lactation. As the promoter of leptin regulates the expression of leptin, polymorphisms at this region could play an important role in the found differences in plasma leptin levels during late pregnancy. Therefore in Chapter 7 we sequenced the leptin promoter and analyzed the sequence for transcription factor binding sites and polymorphisms. All genotyped polymorphisms were associated with prepartum leptin concentrations but in Chapter 8 we showed that three polymorphisms located on a 135 bp promoter region (282 to 147 bp before the transcription start site) seemed to be important for differences in leptin concentrations during late pregnancy. Also significant differences during the lactation period were found between the two genotype combinations with the highest en lowest leptin concentrations. We proposed a putative pregnancy-dependent enhancer to be located at this site on the leptin promoter.In conclusion, polymorphisms in the leptin gene and its promoter region are associated with traits in dairy cattle like milk yield (RFLP1), energy balance (SNP -963) and fertility (SNP -1457). Further experiments and calculations are needed to investigate if a combination of these three polymorphisms would be useful in future selection. On the promoter region a pregnancy-dependent enhancer might be located. A combination of polymorphisms located at this region might be useful to investigate the function of high leptin levels during pregnancy.