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Department of Animal Science, Cornell University, Ithaca, NY 14853
Corresponding author: Y. R. Boisclair; e-mail: yrb1{at}cornell.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: leptin • nutrition • puberty • development
Abbreviation key: ADG = average daily gain, CLA = conjugated linoleic acids, GH = growth hormone, H = high plane of nutrition, L = low plane of nutrition, RIA = radioimmunoassay, WAT = white adipose tissue
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The regulation and roles of plasma leptin have been studied predominantly in adult animals, and almost always in the context of diseases such as obesity or diabetes (Ahima and Flier, 2000; Ingvartsen and Boisclair, 2001; Spiegelman and Flier, 2001). Very little information is available in early postnatal life in any animals. This information would be particularly useful in growing dairy cattle because variations in the plane of nutrition impact developmental processes that could be regulated by leptin. For example, high planes of nutrition lead to earlier onset of puberty and younger age at calving (Foster and Nagatani, 1999; Sejrsen et al., 2000). In prepubertal heifers, however, the use of this nutritional strategy has also been associated with decreased parenchymal mammary development and decreased future milk yield (Sejrsen et al., 2000). Cues mediating these effects of nutrition remain unknown, but leptin is an attractive candidate given the positive relation between its synthesis and nutrition.
As a first step to evaluate the role of leptin in early postnatal life, we sought to determine the developmental and nutritional regulation of leptin in growing dairy cattle. We also asked whether variation in plasma leptin concentration could explain variation in the onset of puberty in a group of well-fed dairy heifers. Our data indicate that nutrition regulates plasma leptin soon after birth, but that puberty can occur over a wide range of plasma leptin concentrations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immediate postnatal period.
To describe the ontogeny of plasma leptin after birth, a previously described study was used (Diaz et al., 2001). Animals consisted of dairy bulls born in January and February of the same year. They were fed 3.7 L of colostrum at birth and transported to the Cornell Teaching and Research facility within the next 24 h. Upon arrival, calves were fed a colostrum supplement (Lifeline, American Protein Corp., Ames, IA), administered vitamin B complex (The Butler Co., Dublin, OH), selenium, and vitamin E (BoSe, Schering-Plough Animal Health Corp., Union, NJ), elemental iron (The Butler Co.) and vaccinated according to the research protocol (TSV-2, Pfizer Animal Health, Exton, PA; Once PMH and BRSV Vac 4, Bayer Corp., Shawnee Mission, KS).
After 3 to 5 d of adjustment to housing conditions, calves were randomly allocated to one of nine treatments arranged in a 3 x 3 factorial (six animals per treatment). The first factor consisted of plane of nutrition targeted to sustain average daily gains (ADG) of 0.50 (low), 0.95 (medium), or 1.4 kg/d (high). All animals were fed appropriate amounts of a single reconstituted milk replacer (300 g of CP, 200 g of fat, and approximately 440 g of carbohydrate and 60 g of ash per kg DM; Milk Specialties Company, Dundee, IL). Daily allowances were fed in three equal meals at 0700, 1400 and 2100 h; water was available at all times. The second factor consisted of weight at slaughter (65, 85, and 105 kg). For the purpose of the present study, analysis was restricted to three nutrition x BW combinations [Low-65 kg (L-65); Low-105 kg, (L-105); and High-105 kg, (H-105)]. This arrangement allowed the comparison of the low (L) and high (H) planes of nutrition at the same body size (L-105 vs. H-105) and at a comparable chronological age (L-65 vs. H-105). Weekly blood samples were obtained by jugular venipuncture between 1100 and 1300 h. Plasma was prepared immediately and frozen at -20°C until analyzed. Calves were killed by captive bolt stunning and exsanguination when they reached their target weight (between early March and late May).
Ruminant growth phase.
A second study was used to describe the ontogeny of plasma leptin in dairy heifers from shortly after weaning until puberty. All animals were born between January and March of the same year. They were brought to the Cornell Teaching and Research facility at 3 to 5 m of age, and acclimatized for a period of 3 wk. During that time, they were fed a silage-based TMR ad libitum and were vaccinated against infectious bovine rhinotracheitis, parainfluenza type 3, bovine viral diarrhea, and bovine respiratory syncytial viruses (Bovishield 4, Pfizer Animal Health), clostridial diseases (Ultrabac 7, Pfizer Animal Health), and Pastuerella pneumonia (One Shot, Pfizer Animal Health). Vaccination was repeated 4 wk later.
Twelve animals were randomly allocated to a TMR containing 2.6% of a calcium salt of palm fatty acids (control; Ener GII, Bioproducts Inc., Fairlawn, OH) or 2.8% of a calcium salt of conjugated linoleic acid (CLA; Natural Lipids, Hovdebygda, Norway). Each treatment was designed to provide equal amounts of fatty acids. The control fat supplement contained 85% fat and 8.5% Ca, with palmitic, oleic, and linoleic fatty acids accounting for 46, 38, and 11% of total fatty acids, respectively. The CLA supplement contained 75% fat and 17% Ca, with CLA isomers accounting for 39% of the fatty acids; the most abundant CLA isomers present were trans-10, cis-12, and cis-9, trans-11 CLA (28 and 23% of total CLA, respectively). Diets were formulated using the Cornell Net Carbohydrate and Protein System to provide sufficient metabolizable energy and metabolizable protein to sustain a daily gain of 1 kg/d (CNCPS v. 4.0; Fox et al., 2000). Heifers were weighed weekly, and daily feed allowances were adjusted to sustain gains of 1 kg/d.
Blood samples were obtained via jugular venipuncture into evacuated blood collection tubes containing sodium heparin on a weekly basis prior to 225 kg of BW, and twice weekly thereafter. Plasma was prepared immediately and frozen at -20°C until analyzed. Individual animals were declared pubertal when the plasma concentration of progesterone exceeded 1 ng/ml in two consecutive samples. Animals were killed during diestrus of their third estrous cycle by captive bolt stunning and exsanguination.
Analysis of Body Composition
Animals were partitioned into four components representing blood and organs; head, hide, feet, and tail; liver; and carcass (Diaz et al., 2001). Blood and organs comprised kidneys, spleen, pancreas, bladder, reproductive tract, heart-lungs-trachea, blood, and digestive tract (rumen, reticulum, omasum, abomasum, large, and small intestine). The digestive tract was emptied of digesta before inclusion in blood and organs. Weights of all four components were recorded before they were ground separately; however, in the ruminant growth phase study, the liver was included with the blood and organs fraction. Representative samples of each component were freeze-dried and stored until measurement of fat by ether extraction as described previously (Diaz et al., 2001).
Analysis of Metabolites and Hormones
Plasma glucose was measured by the glucose oxidase method and NEFA by the Acyl-CoA synthetase/oxidase method (Block et al., 2001). Plasma leptin was assayed by a double antibody bovine radioimmunoassay (RIA) (Ehrhardt et al., 2000; Block et al., 2001). Plasma concentrations of progesterone, insulin, growth hormone (GH), and IGF-I were measured by specific RIA established in our laboratory (Boisclair et al., 1994; Beam and Butler, 1997); inter and intraassay CV for all assays averaged less than 8 and 10%, respectively.
Statistical Analysis
All data were analyzed using the mixed model procedure of SAS (SAS Institute, Cary, NC). In both studies, changes over time were assessed using a mixed model accounting for time and treatment as the fixed effects, and animal as the random effect. When considering fixed end points (slaughter or puberty), data were analyzed with a mixed model with treatment as the fixed effect (i.e., L-105, L-65, and H-105; control or CLA) and animal as the random effect. Relations between the plasma concentration of leptin and indices of body composition, plasma metabolites, or age were assessed by linear regression.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The profile of plasma leptin diverged for the first time at 3 wk of age when plasma leptin started to rise in the H-105 group (treatment x time; P < 0.001). Plasma leptin concentration continued to increase over time in the H-105 calves, whereas it decreased in the L-105 kg calves until it rebounded above 2 ng/ml during the last 2 wk.
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and 2; P < 0.001). The H-105 calves were nearly twice as fat as the L-105 (Figure 2; P < 0.001), but because of their faster rate of gain, were also younger than the L-105 calves (48 ± 5 vs. 94 ± 3 d; P < 0.01). To compare the effect of nutrition on a chronological basis, plasma leptin and body fat were measured in a group of animals receiving the low plane of nutrition and slaughtered at 65 kg (L-65), when about the same age as the H-105 calves (39 ± 5 d). Plasma leptin and body fat of L-65 calves were identical to those of L-105, and again lower than those of H-105 (Figure 2). These data suggest that plasma leptin fails to increase in the L calves over time because their body fat content is static.
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; P < 0.01) and tended to be correlated positively with plasma glucose (P < 0.07). The concentration of plasma leptin was not correlated with the plasma concentrations of NEFA or GH (data not shown).
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). Over the first 20 wk of treatment, CLA had no effect on plasma leptin or GH but caused a small, consistent decrease in plasma insulin (P < 0.05, Figure 4). Similar results were obtained after 20 wk of treatment, when the slaughter of pubertal animals caused a progressive dwindling in the number of observations (results not shown).
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). At slaughter, plasma leptin concentration was positively correlated with percentage of fat in the carcass, total kilograms of fat and final BW (Figure 6, P 
0.01), but not with plasma GH or insulin (results not shown). These data suggest that the curvilinear relationship between plasma leptin and age reflects an increased rate of fat deposition as animals approach mature size (Figure 5).
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). Moreover, the plasma concentration of leptin was significantly lower in the early puberty than in the late puberty group (Table 1, P < 0.001), whereas the plasma concentrations of insulin and GH did not differ.
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). The plasma concentration of leptin was nearly constant around puberty in the early puberty heifers, whereas it increased in the late puberty heifers (age at puberty x time, P < 0.0001). Moreover, when analyzed at the age when puberty occurred in the early puberty group (d 286), BW, mean plasma leptin, and the profile of plasma leptin of the late puberty heifers were identical to those of early puberty heifers (late puberty at 286 d vs. early puberty, Table 1 and Figure 7). These data indicate that failure to reach puberty early is not explained by a deficit in any of these variables. Overall, these data suggest that plasma leptin does not determine the timing of puberty.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effect of nutrition on circulating leptin during fetal life has not been studied in cattle, but data in sheep suggest that this is unlikely to occur. In that species, the concentration of leptin in fetal plasma is not regulated by moderate variations in maternal nutrition (±50% of requirements), even when imposed during late gestation (Ehrhardt et al., 2002; Mühlhäusler et al., 2002; Yuen et al., 2002). This lack of regulation likely reflects the low adiposity of fetal ruminants (Alexander, 1978; Diaz et al., 2001), and the significant production of leptin by nonadipose tissue where synthesis may not be nutritionally regulated (Ehrhardt et al., 2002). Our data indicate that, in cattle, this state of nonresponsiveness extends to the first 2 wk of postnatal life. By the third week, however, plasma leptin started to rise in male calves receiving the high plane of nutrition. This actually corresponds to the time at which these animals display a significant elevation in fatness relative to birth (Diaz et al., 2001). In contrast, male calves fed according to currently accepted standards (i.e., L calves gaining 0.57 kg/d; NRC 2001) had static adiposity and plasma leptin concentration. We anticipate that female calves would yield similar results, given that the pattern of growth this early in life is virtually the same for both sexes (Kuehn et al., 1994). The same may be true in sheep because conventionally fed lambs (unrestricted suckling and access to a dry diet) have constant plasma leptin between d 19 and 47 of postnatal life (McFadin et al., 2002), whereas lambs fed on a high plane of nutrition have two to threefold higher levels of plasma leptin than those on a low plane of nutrition (Ehrhardt et al., 2001). Overall, our data indicate that plasma leptin in dairy cattle can be increased by nutrition in early postnatal life, but that currently recommended feeding standards do not support increased adiposity and the associated rise in plasma leptin.
The ruminant phase of growth was studied in heifers offered either a conventional or a CLA-enriched fat supplement. During the acclimatization phase of this experiment, they were fed according to recommended standards, and their concentration of plasma leptin averaged 2.2 ng/ml. Chronic CLA supplementation had no effects on growth, adiposity, or fat accumulation, and not surprisingly, no effect on plasma leptin. The absence of CLA effects was not totally unexpected because a substantial portion must have been destroyed by rumen fermentation and because attenuation of lipogenesis requires a higher dose of CLA in WAT than in the mammary gland, as recently discussed by Baumgard et al. (2002). Nevertheless, this general lack of effect provided the opportunity to study the ontogeny of plasma leptin until puberty. Plasma leptin remained nearly constant until d 300, which, developmentally, corresponds to the time when deposition of lean tissue slows down and nutrients become increasingly partitioned towards lipogenesis. These results suggest that, as shown in the preruminant calves, rapidly rising concentrations of plasma leptin are most obvious when the rate of fat accretion increases.
In many mammals, including cattle, undernutrition is a potent negative modulator of reproductive activity (Foster and Nagatani, 1999; Spicer, 2001; Williams et al., 2002). In the growing animal, suboptimal nutrition delays growth, the acquisition of adequate energy stores and puberty (Foster and Nagatani, 1999). Moreover, leptin-deficient mice and humans are in a permanent prepubertal state, and leptin therapy alone is sufficient for resumption and completion of sexual development (Chehab et al., 1996; Mounzih et al., 1997; Farooqi et al., 1999). These observations have led to the hypothesis that leptin could be a primary signal triggering puberty (Foster and Nagatani, 1999). This model predicts that peripubertal animals should have higher plasma leptin than nonpubertal animals. Our data do not support this model because puberty occurred over a range of plasma leptin concentrations and equally well in the presence of static or increasing peripubertal plasma leptin concentrations. The peripubertal rise in plasma leptin reported by others probably reflects fat deposition (Diaz-Torga et al., 2001; Garcia et al., 2002) but is obviously not a mandatory condition for attainment of puberty in dairy cattle. In this context, the report by Diaz-Torga et al. (2001) should be viewed cautiously because plasma concentrations were assayed by a commercial RIA and were 15 to 30 times higher than those measured in ruminants using extensively validated homologous RIA (Delavaud et al., 2000; Ehrhardt et al., 2000). Finally, peripheral leptin administration can increase LH pulsation in underfed, sexually mature sheep but does not trigger its appearance in prepubertal lambs (Jackson et al., 2002; Morrison et al., 2002). Therefore, a rise in plasma leptin is not the developmental event triggering the onset of puberty in ruminants. A similar conclusion has been reached in the rhesus monkey (Plant, 2001), and in mice and rats where puberty also occurs over a range of age and adiposity, and in the absence of any peripubertal change in plasma leptin (Devaskar et al., 1997; Bronson, 2001; Cheung et al., 2001; Engelbregt et al., 2001; Watanobe and Schioth, 2002).
An alternative hypothesis is that leptin is permissive to the occurrence of puberty. In this model, depressed plasma leptin, due to insufficient nutrient availability or depleted body reserve, attenuates the ability of other signals to trigger puberty, or even acts as an absolute gating mechanism (Cunningham et al., 1999). Consistent with this idea, leptin administration corrects the delay in puberty observed in feed-restricted rodents (Cheung et al., 1997, 2001). The minimally effective leptin concentration allowing puberty to occur has not been defined in any species, but appears to be quite low. As shown in this paper, plasma levels of 2.6 ng/ml are sufficient for the occurrence of puberty in cattle. Similarly, timing of puberty is normal in lipoatropic humans, which have virtually no adipose tissue and plasma leptin levels below 1 ng/ml (Pardini et al., 1998; Andreelli et al., 2000).
Insulin is an important positive regulator of leptin synthesis in mature dairy cattle, as shown by positive correlations between their plasma levels, and by elevated plasma leptin during euglycemic-hyperinsulinemic conditions (Block et al., 2001, 2003; Leury et al., 2003). We confirmed the positive effects of insulin in the immediate postnatal period, but we failed to identify this relationship in pubertal heifers. We postulate that the latter reflects the lack of statistical power (narrow range of plasma leptin and insufficient number of animals). Nevertheless, this observation warrants investigations of the ability of insulin to regulate plasma leptin during the ruminant phase of growth in dairy cattle.
In prepubertal heifers, growth rates in excess of 700 g/d have been associated with decreased mammary parenchymal growth, and with decreased milk yield during the first lactation (Radcliff et al., 2000; Sejrsen et al., 2000). In MAC-T cells, a SV-40 transformed mammary epithelial cell line derived from a lactating dairy cow (Huynh et al., 1991), leptin caused a 20% reduction in IGF-I stimulated thymidine incorporation (Silva et al., 2002). These observations have led to the idea that increased plasma leptin could be responsible for mediating the negative effects of nutrition on prepubertal mammary development. However, our data showing that ruminant heifers growing at ~1 kg/d maintain low and nearly constant level of plasma leptin during most of the prepubertal period are somewhat inconsistent with this idea. Moreover, negative effects of leptin on IGF-I mediated proliferation could not be demonstrated in primary epithelial cells isolated from prepubertal heifers (Purup and Sejrsen, 2000). However, it remains possible that mammary development was depressed in our study, and that local synthesis rather than plasma delivery is the primary determinant of leptin concentration in the vicinity of the developing mammary epithelium. In this context, leptin mRNA is readily detected in mammary WAT of growing dairy heifers (Block and Boisclair, unpublished results), but whether nutrition regulates leptin synthesis in mammary WAT remains to be shown.
In summary, plasma leptin can be increased under conditions leading to accelerated fat deposition in growing dairy cattle (i.e., higher plane of nutrition after birth, age-dependent shift in nutrient partitioning). An age-dependent increase in plasma leptin is not required for onset of puberty, but it remains possible that leptin modulates mammary parenchymal development.
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FOOTNOTES |
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Received for publication January 20, 2003. Accepted for publication April 4, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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