- Email: [email protected]
Leptin, insulin and ghrelin levels in goat milk and in plasma of suckling kids
Small Ruminant Research 79 (2008) 38–41
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Leptin, insulin and ghrelin levels in goat milk and in plasma of suckling kids夽 D. Magistrelli ∗ , G. Polo Dimel, F. Rosi Department of Animal Science, University of Milan, via G. Celoria 2, Milan 20133, Italy
a r t i c l e
i n f o
Article history: Available online 15 August 2008 Keywords: Leptin Insulin Ghrelin Goat milk Bioactive peptides
a b s t r a c t Milk is not just the basic source of nourishment for the neonate, but it is also a vector of bioactive peptides that play a possible role in neonatal development. For the increasing utilization of goat milk in infant formulas for human nutrition, it could be of interest to quantify the levels of leptin, insulin and ghrelin in goat milk and in plasma of suckling goat kids and to evaluate the relationship between ingested and circulating levels of hormones in growing animals. For this purpose, 10 suckling kids were observed from the 3rd to the 5th week of age (before the beginning of the weaning period). During the experimental period, body weight and milk intake were recorded. Milk and blood samples were taken before the first meal of the day on week 3, 4 and 5. Plasma and milk were analysed for leptin, insulin and ghrelin. As expected, body weight and milk intake increased during the experimental period. Although daily intake of hormones and growth factors with milk increased, plasma leptin level of suckling kids decreased (P = 0.02). On the contrary, plasma level of insulin increased more than four times (P < 0.01) and was highly correlated (P < 0.01) to the daily quantity of hormone ingested. This trend was probably associated to the increased milk intake, but it is even possible to hypothesize the presence of receptors that facilitate the migration of milk-borne insulin through the gastrointestinal mucosa of the suckling kids. This hypothesis is supported by the fundamental role played by insulin in the functional development of pancreas; in fact in rats plasma insulin level arises just before weaning and this is supposed to stimulate pancreatic ␣-amylase gene expression. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Milk is the basic source of nourishment, containing specific proteins in colloidal dispersion, lipids in emulsified globules coated by membrane, minerals and lactose in true solution, and providing all the elements required for neonatal growth (Jensen et al., 1995). Milk is also a vector of
夽 This paper is part of the special issue entitled 5th International Symposium on The Challenge to Sheep and Goats Milk Sectors Guest Edited by Antonio Pirisi, André Ayerbe, Giovanni Piredda, George Psathas and Yvette Soustre. ∗ Corresponding author. Tel.: +39 0250316443; fax: +39 0250316434. E-mail address: [email protected] (D. Magistrelli). 0921-4488/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2008.07.004
bioactive peptides. The finding of hormones in milk was described as early as 1929 (Yaida, 1929) and, to date, many growth factors have been identified (Grosvenor et al., 1993), even in concentration exceeding that of maternal plasma (Rosi and Rapetti, 2004; Magistrelli et al., 2005). Some bioactive molecules are transported (unchanged in structure and activity) into milk from the maternal blood; others are synthesized by the mammary gland and posttranslationally modified to change their biological activity (Campana and Baumrucker, 1995). Interestingly, some hormones (i.e., thyroid hormones, estrogens, prolactin, etc.) and non-hormonal bioactive substances (i.e., lactoferrin, transferrin, etc.) appear both to be synthesized by the mammary tissue of various species and also transported from maternal circulation (Campana and Baumrucker, 1995).
D. Magistrelli et al. / Small Ruminant Research 79 (2008) 38–41
Milk-borne peptides can pass across the gastrointestinal mucosa (Gonnella et al., 1987) and enter the systemic circulation via a receptor-mediated process (Xu et al., 2000; Blum and Baumrucker, 2002), playing a possible role in accomplishing the immature ability of the suckling animals to produce hormones and growth factors (Kinouchi et al., 2000). Among milk bioactive compounds there are leptin, insulin and ghrelin. All these peptides are involved in the regulation of food intake. Leptin is the product of the ob gene and it is mainly secreted by adipose cells (Houseknecht et al., 1998). Circulating leptin has a 147 amino-acids structure and targets the hypothalamus to coordinate food intake. Leptin acts on peripheral tissue to promote energy expenditure and nutrient utilization (Ahima and Flier, 2000). Insulin is a 5.8-kDa protein synthesized in the pancreatic -cells and secreted in response of elevation of plasma glucose level. Insulin is responsible for glucose uptake from the blood stream into adipose, muscular and hepatic cells (Pessin and Saltiel, 2000), but it also modulates peripheral satiety signals and directly targets the central nervous system to inhibit food intake (Gale et al., 2004). By contrast, ghrelin is a 28 amino-acids acylated peptide from stomach, which plays a role in stimulating GH release by the pituitary gland and in affecting feeding behaviour as stimulator of food intake (Kojima and Kangawa, 2005). In order to understand the relationship between ingested and circulating levels of hormones in growing animal, the aim of this study was to quantify the level of leptin, insulin and ghrelin in goat milk and in plasma of suckling goat kids. Moreover, it could be important to evaluate the presence of bioactive peptides in goat milk, because this product is widely consumed in Mediterranean and Middle East countries (Grant et al., 2005). In particular, even if data on the hypo-allergenicity, tolerability and safety of goat milk are conflicting (Wilson and Hamburger, 1988; Park, 1994; Biggart, 1996; Bellioni Businco et al., 1999), goat milk – modified and fortified to meet infant formula regulations – is used as an appropriate alternative for infants who cannot be breast-fed (Grant et al., 2005). 2. Materials and methods Immediately after birth, 10 Saanen goat kids were separated from their mother and housed in a steel box measuring 3.0 m × 4.5 m (l × d) with straw bedstead. All kids were manually fed colostrum of the respective mother and then goat milk, until the 5th week of age, when kids started a weaning protocol. The milk administered to the kids derived from the massive milk obtained from all the mothers. During the entire experimental period, goat kids were fed twice a day, at 9:00 and 19:00. From day 4 to week 5 of age, milk intake was recorded daily and body weight was recorded weekly. On week 3, 4 and 5 of age, jugular vein blood samples were taken before the first meal of the day. After the withdrawal, blood was immediately centrifuged at 2000 × g for 15 min and then stored at −20 ◦ C until analysis. On the same days, goat milk samples were also taken and stored at −20 ◦ C. At the end of the experimental period, morning and evening milk (collected during the same day) were mixed and an aliquot was analysed for dry matter by drying at 102 ◦ C (IDF, 1987), crude protein by Kjeldahl method (IDF, 1986), fat by Röse-Gottlieb method (IDF, 1996) and lactose content by mid-IR spectrophotometric method (IDF, 1990). Another aliquot was sonicated and centrifuged at 2150 × g for 30 min at 4 ◦ C. Plasma
39
Table 1 Goat milk composition between 3 and 5 weeks of lactation
Dry matter Protein Fat Lactose
% as fed
S.D.
% Dry matter
S.D.
12.2 3.25 3.61 4.72
0.75 0.09 0.57 0.12
26.6 29.5 38.6
0.72 4.67 0.96
and sonicated-centrifuged milk were tested for leptin (Multispecies Leptin RIA Kit, Linco Research Inc, St. Charles, MO, USA) and insulin (Insik-5, Dia Sorin Spa, Saluggia, VC, Italy) by radio-immuno assays and ghrelin (Total Ghrelin, Diagnostics System Laboratories Inc., Webster, TX, USA) by ELISA method. Data were analysed by the ANOVA procedure of (SAS, 1999) and correlations expressed by Pearson coefficient.
3. Results and discussion Goat milk composition and milk insulin, leptin and ghrelin levels are reported in Tables 1 and 2, respectively. As expressed in Figs. 1 and 2, body weight and milk intake increased (P < 0.01) during the experimental period. Although, during the experimental period, daily intake of hormones and growth factors with milk increased, plasma leptin level of suckling kids decreased (P = 0.02) (Fig. 3). This result is in accordance with findings in other species. For example, in rats plasma leptin is observed to be at the highest level during the first hours after birth and associated to the high expression of the ob gene in brown adipose tissue (Dessolin et al., 1997). In these animals, the high concentration of leptin at birth may be favourable for increasing heat production in brown adipose tissue during non-shivering thermogenesis for reaction to cold stress (Dessolin et al., 1997). In humans too, plasma leptin concentration rapidly declines during the neonatal period (Yokota, 2003) and until to 4 weeks of age (Helland et al., 1998). After that, Helland et al. (1998) observed an increase in plasma leptin concentration until to 14 weeks after birth. The experimental period of the present study was too shirt to detect an eventual later arise in plasma leptin level. Anyway, in postweaning goats plasma leptin peaks at the onset of puberty (Vitali et al., 2005). As shown in Fig. 3, plasma ghrelin did not vary, during our experimental period. In infancy, ghrelin may exert a strong growth hormone releasing action primarily promoting body growth (Savino et al., 2006). In humans, ghrelin level increases after birth peaking during the first 2 years of life, then decreases until the end of puberty to reach the adult level (Soriano Guillen et al., 2004). In rats gastric expression of ghrelin increases in an age-dependent manner from neonatal to adult stages (Hayashida et al., 2002). The discrepancy with our data suggests that it has to be clarified whether the regulation of ghrelin secretion in suckling goat kids is similar to that of other species. This assumption is supported by Kobayashi et al. (2006), Table 2 Growth factors in milk between 3 and 5 weeks of lactation
Leptin Insulin Ghrelin
mmol/l
S.D.
0.53 0.30 1.01
0.04 0.06 0.04
40
D. Magistrelli et al. / Small Ruminant Research 79 (2008) 38–41
Fig. 1. Body weight (mean value ± S.D.).
who recently observed that plasma ghrelin concentration in goats was not changed by feeding or weaning, although feeding reduces plasma ghrelin in various animal species (Hayashida et al., 2001; Tschop et al., 2001). On the contrary, during the 3 weeks experimental period, plasma level of insulin increased more than 4 times (P < 0.01) (Fig. 3) and insulin level was significantly correlated to the daily quantity of hormone ingested (P < 0.01). This latter datum was probably due to the increasing amount of energy intake during the experimental period. However, milk may be considered a source of insulin for the sucklings. Kinouchi et al. (1998) observed that plasma insulin level in rats increased in the early stage of weaning, and insulin administration with milk enforced this increase. Moreover, they observed a close relationship between plasma insulin level and pancreatic ␣-amylase activity, suggesting the involvement of insulin as a stimulatory factor of pancreatic functional development (Kinouchi et al., 1998). For this reason, it is possible to hypothesize that milkborne insulin can cross the gastrointestinal mucosa and enter the systemic circulation of suckling kids, by specific receptors, inducing the increase in basal level of insulin. 4. Conclusions
Fig. 2. Individual daily milk intake (mean value ± S.D.).
Milk-borne leptin and ghrelin do not seem to affect circulating levels of hormones in the sucklings. Oppositely, insulin could be absorbed from milk and enter the systemic circulation, increasing its plasmatic level in suckling kids. This suggests a possible role for milk-borne insulin in the regulation of endocrine and digestive function in pre-weaning kids. Further studies are needed in order to verify this hypothesis and clarify the possible effect of milk peptides on the development of young animals. Acknowledgements Special thanks to Prof. Luca Rapetti and Mr. Davide De Angeli for the help in livestock practice, during the entire experimental period. References
Fig. 3. Growth factors in plasma (mean value ± S.D.).
Ahima, R.S., Flier, J.S., 2000. Leptin. Physiol. Rev. 62, 413–437. Bellioni Businco, B., Paganelli, R., Lucenti, P., Giampietro, P.G., Perborn, H., Businco, L., 1999. Allergenicity of goat’s milk in children with cow’s milk allergy. J. Allergy Clin. Immun. 103, 1191–1194. Biggart, T., 1996. Goat milk for the allergic child. Paediatr. Today 4, 37–39. Blum, J.W., Baumrucker, C.R., 2002. Colostral and milk insulin-like growth factors and related substances: mammary gland and neonatal (intestinal and systemic) targets. Domest. Anim. Endocrin. 23, 101–110. Campana, W.M., Baumrucker, C.R., 1995. Hormones and growth factors in bovine milk. In: Jensen, R.G. (Ed.), Handbook of Milk Composition. Academic Press, San Diego, CA, pp. 476–494. Dessolin, S., Schalling, M., Champigny, O., Lönnqvist, F., Ailhaud, G., Dani, C., Ricquier, D., 1997. Leptin gene is expressed in rat brown adipose tissue at birth. FASEB J. 11, 382–387. Gale, S.M., Castracane, V.D., Mantzoros, C.S., 2004. Energy homeostasis, obesity and eating disorders: recent advances in endocrinology. J. Nutr. 134, 295–298. Gonnella, P.A., Siminoski, K., Murphy, R.A., Neutra, M.R., 1987. Transepithelial transport of epidermal growth factor by absorptive cells in suckling rat ileum. J. Clin. Invest. 80, 22–32. Grant, C., Rotherham, B., Sharpe, S., Scragg, R., Thompson, J., Andrews, J., Wall, C., Murphy, J., Lowry, D., 2005. Randomized, double-blind com-
D. Magistrelli et al. / Small Ruminant Research 79 (2008) 38–41 parison of growth in infants receiving goat milk formula versus cow milk infant formula. J. Paediatr. Child. Health 41, 564–568. Grosvenor, C.E., Picciano, M.F., Baumrucker, C.R., 1993. Hormones and growth factors in milk. Endocr. Rev. 14, 710–728. Hayashida, T., Murakami, K., Mogi, K., Nishihara, M., Nakazato, M., Mondal, M.S., Horii, Y., Kojima, M., Kangawa, K., Murakami, N., 2001. Ghrelin in domestic animals: distribution in stomach and its possible role. Dom. Anim. Endocrinol. 21, 17–24. Hayashida, T., Nakahara, K., Mondal, M.S., Date, Y., Nakazato, M., Kojima, M., Kangawa, K., Murakami, N., 2002. Ghrelin in neonatal rats: distribution in stomach and its possible role. J. Endocrinol. 173, 239–245. Helland, I.B., Reseland, J.E., Saugstad, O.D., Drevon, C.A., 1998. Leptin levels in pregnant women and newborn infants: gender differences and reduction during the neonatal period. Pediatrics 101, 12–16. Houseknecht, K.L., Baile, C.A., Matteri, R.L., Spurlock, M.E., 1998. The biology of leptin: a review. J. Anim. Sci. 76, 1405–1420. International Dairy Federation, 1986. Milk: Determination of Nitrogen Content and Calculation of Crude Protein Content—Kjeldahl Method (reference method) FIL-IDF Standard 20A:1986. Brussels, Belgium. International Dairy Federation, 1987. Milk Cream and Evaporated Milk: Determination of Total Solids Content—FIL-IDF Standard 021B:1987. Brussels, Belgium. International Dairy Federation, 1990. Milk: Determination of Fat Protein and Lactose Content—Mid-IR Method (reference method). FIL-IDF Standard 141A:1990. Brussels, Belgium. International Dairy Federation, 1996. Milk: Determination of Fat Content—Röse-Gottlieb Gravimetric Method (reference method) FILIDF Standard 1D:1996. Brussels, Belgium. Jensen, R.G., Couch, S.C., Hansen, J.W., Lien, E.L., Ostrom, K.M., Bracco, U., Clemens, R.A., 1995. Infant formulas. In: Jensen, R.G. (Ed.), Handbook of Milk Composition. Academic Press, San Diego, CA, pp. 835–856. Kinouchi, T., Koizumi, K., Kuwata, T., Yajima, T., 1998. Crucial role of milkborne insulin in the development of pancreatic amylase at the onset of weaning in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 275, 1958–1967. Kinouchi, T., Koizumi, K., Kuwata, T., Yajima, T., 2000. Milk-borne insulin with trypsin inhibitor in milk induces pancreatic amylase development at the onset of weaning in rats. J. Pediatr. Gastr. Nutr. 30, 515–521. Kobayashi, Y., Onodera, M., Yoshida, M., Sugino, T., Katoh, K., Obara, Y., 2006. Postprandial changes in plasma concentrations of growth hormone and ghrelin around weaning in the goat. Gen. Comp. Endocr. 148, 368–374.
41
Kojima, M., Kangawa, K., 2005. Ghrelin: structure and function. Physiol. Rev. 85, 495–522. Magistrelli, D., Valli, A., Rosi, F., 2005. Insulin and IGF-1 in goat milk: influence of the diet. In: Proceedings of the ASPA 16th Congress, Turin (Italy), June 28–30, 2005, It. J. Anim. Sci. (4 Suppl. 2), pp. 386–388. Park, Y.W., 1994. Hypoallergenic and therapeutic significance of goat milk. Small Rumin. Res. 14, 151–159. Pessin, J.E., Saltiel, A.R., 2000. Signaling pathways in insulin action: molecular targets of insulin resistance. J. Clin. Invest. 106, 165–169. Rosi, F., Rapetti, L., 2004. Bioactive components in goat milk and plasma. In: Proceedings of the Xth International Symposium on Ruminant Physiology, Copenhagen (Denmark), August 30–September 4, 2004. J. Anim. Feed Sci. (13 Suppl. 1), pp. 701–704. Savino, F., Grassino, E.C., Fissore, M.F., Guidi, C., Liguori, S.A., Silvestro, L., Oggero, R., Miniero, R., 2006. Ghrelin, motilin, insulin concentration in healthy infants in the first months of life: relation to fasting time and anthropometry. Clin. Endocrinol. 65, 158–162. Soriano Guillen, L., Barrios, V., Chowen, J.A., Sanchez, I., Vila, S., Quero, J., Argente, J., 2004. Ghrelin levels from fetal life through early adulthood: relationship with endocrine and metabolic and anthropometric measures. J. Pediatr. 144, 30–35. SAS, 1999. User’s Guide: Statistics Version 7.03. SAS Institute Inc., Cary, NC, USA. Tschop, M., Wawarta, R., Riepl, R.L., Friedrich, S., Bidlingmaier, M., Landgraf, R., Folwaczny, C., 2001. Post-prandial decrease of circulating human ghrelin levels. J. Endocrinol. Invest. 24, RC19–RC21. Vitali, A., Magistrelli, D., Azevedo, J., Bernabucci, U., Ronchi, B., Rosi, F., 2005. Leptin and puberty in goat. In: Proceedings of the ASPA 16th Congress, Turin (Italy), June 28–30, 2005. It. J. Anim. Sci. (4 Suppl. 2), pp. 383–385. Wilson, N.W., Hamburger, R.N., 1988. Allergy to cow’s milk in the first year of life and its prevention. Ann. Allergy 61, 323–327. Xu, R.J., Wang, F., Zhang, S.H., 2000. Postnatal adaptation of the gastrointestinal tract in neonatal pigs: a possible role of milk-borne growth factors. Livest. Prod. Sci. 66, 95–107. Yaida, N., 1929. Ovarian hormone in blood of pregnant women: ovarian hormone in urine of pregnant women: ovarian hormone in milk of pregnant animals. Trans. Jpn. Pathol. Soc. 189, 93–101. Yokota, I., 2003. Roles of leptin and ghrelin during the perinatal period. Clin. Pediatr. Endocrinol. 12, 57–65.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Leptin, insulin and ghrelin levels in goat milk and in plasma of suckling kids夽 D. Magistrelli ∗ , G. Polo Dimel, F. Rosi Department of Animal Science, University of Milan, via G. Celoria 2, Milan 20133, Italy
a r t i c l e
i n f o
Article history: Available online 15 August 2008 Keywords: Leptin Insulin Ghrelin Goat milk Bioactive peptides
a b s t r a c t Milk is not just the basic source of nourishment for the neonate, but it is also a vector of bioactive peptides that play a possible role in neonatal development. For the increasing utilization of goat milk in infant formulas for human nutrition, it could be of interest to quantify the levels of leptin, insulin and ghrelin in goat milk and in plasma of suckling goat kids and to evaluate the relationship between ingested and circulating levels of hormones in growing animals. For this purpose, 10 suckling kids were observed from the 3rd to the 5th week of age (before the beginning of the weaning period). During the experimental period, body weight and milk intake were recorded. Milk and blood samples were taken before the first meal of the day on week 3, 4 and 5. Plasma and milk were analysed for leptin, insulin and ghrelin. As expected, body weight and milk intake increased during the experimental period. Although daily intake of hormones and growth factors with milk increased, plasma leptin level of suckling kids decreased (P = 0.02). On the contrary, plasma level of insulin increased more than four times (P < 0.01) and was highly correlated (P < 0.01) to the daily quantity of hormone ingested. This trend was probably associated to the increased milk intake, but it is even possible to hypothesize the presence of receptors that facilitate the migration of milk-borne insulin through the gastrointestinal mucosa of the suckling kids. This hypothesis is supported by the fundamental role played by insulin in the functional development of pancreas; in fact in rats plasma insulin level arises just before weaning and this is supposed to stimulate pancreatic ␣-amylase gene expression. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Milk is the basic source of nourishment, containing specific proteins in colloidal dispersion, lipids in emulsified globules coated by membrane, minerals and lactose in true solution, and providing all the elements required for neonatal growth (Jensen et al., 1995). Milk is also a vector of
夽 This paper is part of the special issue entitled 5th International Symposium on The Challenge to Sheep and Goats Milk Sectors Guest Edited by Antonio Pirisi, André Ayerbe, Giovanni Piredda, George Psathas and Yvette Soustre. ∗ Corresponding author. Tel.: +39 0250316443; fax: +39 0250316434. E-mail address: [email protected] (D. Magistrelli). 0921-4488/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2008.07.004
bioactive peptides. The finding of hormones in milk was described as early as 1929 (Yaida, 1929) and, to date, many growth factors have been identified (Grosvenor et al., 1993), even in concentration exceeding that of maternal plasma (Rosi and Rapetti, 2004; Magistrelli et al., 2005). Some bioactive molecules are transported (unchanged in structure and activity) into milk from the maternal blood; others are synthesized by the mammary gland and posttranslationally modified to change their biological activity (Campana and Baumrucker, 1995). Interestingly, some hormones (i.e., thyroid hormones, estrogens, prolactin, etc.) and non-hormonal bioactive substances (i.e., lactoferrin, transferrin, etc.) appear both to be synthesized by the mammary tissue of various species and also transported from maternal circulation (Campana and Baumrucker, 1995).
D. Magistrelli et al. / Small Ruminant Research 79 (2008) 38–41
Milk-borne peptides can pass across the gastrointestinal mucosa (Gonnella et al., 1987) and enter the systemic circulation via a receptor-mediated process (Xu et al., 2000; Blum and Baumrucker, 2002), playing a possible role in accomplishing the immature ability of the suckling animals to produce hormones and growth factors (Kinouchi et al., 2000). Among milk bioactive compounds there are leptin, insulin and ghrelin. All these peptides are involved in the regulation of food intake. Leptin is the product of the ob gene and it is mainly secreted by adipose cells (Houseknecht et al., 1998). Circulating leptin has a 147 amino-acids structure and targets the hypothalamus to coordinate food intake. Leptin acts on peripheral tissue to promote energy expenditure and nutrient utilization (Ahima and Flier, 2000). Insulin is a 5.8-kDa protein synthesized in the pancreatic -cells and secreted in response of elevation of plasma glucose level. Insulin is responsible for glucose uptake from the blood stream into adipose, muscular and hepatic cells (Pessin and Saltiel, 2000), but it also modulates peripheral satiety signals and directly targets the central nervous system to inhibit food intake (Gale et al., 2004). By contrast, ghrelin is a 28 amino-acids acylated peptide from stomach, which plays a role in stimulating GH release by the pituitary gland and in affecting feeding behaviour as stimulator of food intake (Kojima and Kangawa, 2005). In order to understand the relationship between ingested and circulating levels of hormones in growing animal, the aim of this study was to quantify the level of leptin, insulin and ghrelin in goat milk and in plasma of suckling goat kids. Moreover, it could be important to evaluate the presence of bioactive peptides in goat milk, because this product is widely consumed in Mediterranean and Middle East countries (Grant et al., 2005). In particular, even if data on the hypo-allergenicity, tolerability and safety of goat milk are conflicting (Wilson and Hamburger, 1988; Park, 1994; Biggart, 1996; Bellioni Businco et al., 1999), goat milk – modified and fortified to meet infant formula regulations – is used as an appropriate alternative for infants who cannot be breast-fed (Grant et al., 2005). 2. Materials and methods Immediately after birth, 10 Saanen goat kids were separated from their mother and housed in a steel box measuring 3.0 m × 4.5 m (l × d) with straw bedstead. All kids were manually fed colostrum of the respective mother and then goat milk, until the 5th week of age, when kids started a weaning protocol. The milk administered to the kids derived from the massive milk obtained from all the mothers. During the entire experimental period, goat kids were fed twice a day, at 9:00 and 19:00. From day 4 to week 5 of age, milk intake was recorded daily and body weight was recorded weekly. On week 3, 4 and 5 of age, jugular vein blood samples were taken before the first meal of the day. After the withdrawal, blood was immediately centrifuged at 2000 × g for 15 min and then stored at −20 ◦ C until analysis. On the same days, goat milk samples were also taken and stored at −20 ◦ C. At the end of the experimental period, morning and evening milk (collected during the same day) were mixed and an aliquot was analysed for dry matter by drying at 102 ◦ C (IDF, 1987), crude protein by Kjeldahl method (IDF, 1986), fat by Röse-Gottlieb method (IDF, 1996) and lactose content by mid-IR spectrophotometric method (IDF, 1990). Another aliquot was sonicated and centrifuged at 2150 × g for 30 min at 4 ◦ C. Plasma
39
Table 1 Goat milk composition between 3 and 5 weeks of lactation
Dry matter Protein Fat Lactose
% as fed
S.D.
% Dry matter
S.D.
12.2 3.25 3.61 4.72
0.75 0.09 0.57 0.12
26.6 29.5 38.6
0.72 4.67 0.96
and sonicated-centrifuged milk were tested for leptin (Multispecies Leptin RIA Kit, Linco Research Inc, St. Charles, MO, USA) and insulin (Insik-5, Dia Sorin Spa, Saluggia, VC, Italy) by radio-immuno assays and ghrelin (Total Ghrelin, Diagnostics System Laboratories Inc., Webster, TX, USA) by ELISA method. Data were analysed by the ANOVA procedure of (SAS, 1999) and correlations expressed by Pearson coefficient.
3. Results and discussion Goat milk composition and milk insulin, leptin and ghrelin levels are reported in Tables 1 and 2, respectively. As expressed in Figs. 1 and 2, body weight and milk intake increased (P < 0.01) during the experimental period. Although, during the experimental period, daily intake of hormones and growth factors with milk increased, plasma leptin level of suckling kids decreased (P = 0.02) (Fig. 3). This result is in accordance with findings in other species. For example, in rats plasma leptin is observed to be at the highest level during the first hours after birth and associated to the high expression of the ob gene in brown adipose tissue (Dessolin et al., 1997). In these animals, the high concentration of leptin at birth may be favourable for increasing heat production in brown adipose tissue during non-shivering thermogenesis for reaction to cold stress (Dessolin et al., 1997). In humans too, plasma leptin concentration rapidly declines during the neonatal period (Yokota, 2003) and until to 4 weeks of age (Helland et al., 1998). After that, Helland et al. (1998) observed an increase in plasma leptin concentration until to 14 weeks after birth. The experimental period of the present study was too shirt to detect an eventual later arise in plasma leptin level. Anyway, in postweaning goats plasma leptin peaks at the onset of puberty (Vitali et al., 2005). As shown in Fig. 3, plasma ghrelin did not vary, during our experimental period. In infancy, ghrelin may exert a strong growth hormone releasing action primarily promoting body growth (Savino et al., 2006). In humans, ghrelin level increases after birth peaking during the first 2 years of life, then decreases until the end of puberty to reach the adult level (Soriano Guillen et al., 2004). In rats gastric expression of ghrelin increases in an age-dependent manner from neonatal to adult stages (Hayashida et al., 2002). The discrepancy with our data suggests that it has to be clarified whether the regulation of ghrelin secretion in suckling goat kids is similar to that of other species. This assumption is supported by Kobayashi et al. (2006), Table 2 Growth factors in milk between 3 and 5 weeks of lactation
Leptin Insulin Ghrelin
mmol/l
S.D.
0.53 0.30 1.01
0.04 0.06 0.04
40
D. Magistrelli et al. / Small Ruminant Research 79 (2008) 38–41
Fig. 1. Body weight (mean value ± S.D.).
who recently observed that plasma ghrelin concentration in goats was not changed by feeding or weaning, although feeding reduces plasma ghrelin in various animal species (Hayashida et al., 2001; Tschop et al., 2001). On the contrary, during the 3 weeks experimental period, plasma level of insulin increased more than 4 times (P < 0.01) (Fig. 3) and insulin level was significantly correlated to the daily quantity of hormone ingested (P < 0.01). This latter datum was probably due to the increasing amount of energy intake during the experimental period. However, milk may be considered a source of insulin for the sucklings. Kinouchi et al. (1998) observed that plasma insulin level in rats increased in the early stage of weaning, and insulin administration with milk enforced this increase. Moreover, they observed a close relationship between plasma insulin level and pancreatic ␣-amylase activity, suggesting the involvement of insulin as a stimulatory factor of pancreatic functional development (Kinouchi et al., 1998). For this reason, it is possible to hypothesize that milkborne insulin can cross the gastrointestinal mucosa and enter the systemic circulation of suckling kids, by specific receptors, inducing the increase in basal level of insulin. 4. Conclusions
Fig. 2. Individual daily milk intake (mean value ± S.D.).
Milk-borne leptin and ghrelin do not seem to affect circulating levels of hormones in the sucklings. Oppositely, insulin could be absorbed from milk and enter the systemic circulation, increasing its plasmatic level in suckling kids. This suggests a possible role for milk-borne insulin in the regulation of endocrine and digestive function in pre-weaning kids. Further studies are needed in order to verify this hypothesis and clarify the possible effect of milk peptides on the development of young animals. Acknowledgements Special thanks to Prof. Luca Rapetti and Mr. Davide De Angeli for the help in livestock practice, during the entire experimental period. References
Fig. 3. Growth factors in plasma (mean value ± S.D.).
Ahima, R.S., Flier, J.S., 2000. Leptin. Physiol. Rev. 62, 413–437. Bellioni Businco, B., Paganelli, R., Lucenti, P., Giampietro, P.G., Perborn, H., Businco, L., 1999. Allergenicity of goat’s milk in children with cow’s milk allergy. J. Allergy Clin. Immun. 103, 1191–1194. Biggart, T., 1996. Goat milk for the allergic child. Paediatr. Today 4, 37–39. Blum, J.W., Baumrucker, C.R., 2002. Colostral and milk insulin-like growth factors and related substances: mammary gland and neonatal (intestinal and systemic) targets. Domest. Anim. Endocrin. 23, 101–110. Campana, W.M., Baumrucker, C.R., 1995. Hormones and growth factors in bovine milk. In: Jensen, R.G. (Ed.), Handbook of Milk Composition. Academic Press, San Diego, CA, pp. 476–494. Dessolin, S., Schalling, M., Champigny, O., Lönnqvist, F., Ailhaud, G., Dani, C., Ricquier, D., 1997. Leptin gene is expressed in rat brown adipose tissue at birth. FASEB J. 11, 382–387. Gale, S.M., Castracane, V.D., Mantzoros, C.S., 2004. Energy homeostasis, obesity and eating disorders: recent advances in endocrinology. J. Nutr. 134, 295–298. Gonnella, P.A., Siminoski, K., Murphy, R.A., Neutra, M.R., 1987. Transepithelial transport of epidermal growth factor by absorptive cells in suckling rat ileum. J. Clin. Invest. 80, 22–32. Grant, C., Rotherham, B., Sharpe, S., Scragg, R., Thompson, J., Andrews, J., Wall, C., Murphy, J., Lowry, D., 2005. Randomized, double-blind com-
D. Magistrelli et al. / Small Ruminant Research 79 (2008) 38–41 parison of growth in infants receiving goat milk formula versus cow milk infant formula. J. Paediatr. Child. Health 41, 564–568. Grosvenor, C.E., Picciano, M.F., Baumrucker, C.R., 1993. Hormones and growth factors in milk. Endocr. Rev. 14, 710–728. Hayashida, T., Murakami, K., Mogi, K., Nishihara, M., Nakazato, M., Mondal, M.S., Horii, Y., Kojima, M., Kangawa, K., Murakami, N., 2001. Ghrelin in domestic animals: distribution in stomach and its possible role. Dom. Anim. Endocrinol. 21, 17–24. Hayashida, T., Nakahara, K., Mondal, M.S., Date, Y., Nakazato, M., Kojima, M., Kangawa, K., Murakami, N., 2002. Ghrelin in neonatal rats: distribution in stomach and its possible role. J. Endocrinol. 173, 239–245. Helland, I.B., Reseland, J.E., Saugstad, O.D., Drevon, C.A., 1998. Leptin levels in pregnant women and newborn infants: gender differences and reduction during the neonatal period. Pediatrics 101, 12–16. Houseknecht, K.L., Baile, C.A., Matteri, R.L., Spurlock, M.E., 1998. The biology of leptin: a review. J. Anim. Sci. 76, 1405–1420. International Dairy Federation, 1986. Milk: Determination of Nitrogen Content and Calculation of Crude Protein Content—Kjeldahl Method (reference method) FIL-IDF Standard 20A:1986. Brussels, Belgium. International Dairy Federation, 1987. Milk Cream and Evaporated Milk: Determination of Total Solids Content—FIL-IDF Standard 021B:1987. Brussels, Belgium. International Dairy Federation, 1990. Milk: Determination of Fat Protein and Lactose Content—Mid-IR Method (reference method). FIL-IDF Standard 141A:1990. Brussels, Belgium. International Dairy Federation, 1996. Milk: Determination of Fat Content—Röse-Gottlieb Gravimetric Method (reference method) FILIDF Standard 1D:1996. Brussels, Belgium. Jensen, R.G., Couch, S.C., Hansen, J.W., Lien, E.L., Ostrom, K.M., Bracco, U., Clemens, R.A., 1995. Infant formulas. In: Jensen, R.G. (Ed.), Handbook of Milk Composition. Academic Press, San Diego, CA, pp. 835–856. Kinouchi, T., Koizumi, K., Kuwata, T., Yajima, T., 1998. Crucial role of milkborne insulin in the development of pancreatic amylase at the onset of weaning in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 275, 1958–1967. Kinouchi, T., Koizumi, K., Kuwata, T., Yajima, T., 2000. Milk-borne insulin with trypsin inhibitor in milk induces pancreatic amylase development at the onset of weaning in rats. J. Pediatr. Gastr. Nutr. 30, 515–521. Kobayashi, Y., Onodera, M., Yoshida, M., Sugino, T., Katoh, K., Obara, Y., 2006. Postprandial changes in plasma concentrations of growth hormone and ghrelin around weaning in the goat. Gen. Comp. Endocr. 148, 368–374.
41
Kojima, M., Kangawa, K., 2005. Ghrelin: structure and function. Physiol. Rev. 85, 495–522. Magistrelli, D., Valli, A., Rosi, F., 2005. Insulin and IGF-1 in goat milk: influence of the diet. In: Proceedings of the ASPA 16th Congress, Turin (Italy), June 28–30, 2005, It. J. Anim. Sci. (4 Suppl. 2), pp. 386–388. Park, Y.W., 1994. Hypoallergenic and therapeutic significance of goat milk. Small Rumin. Res. 14, 151–159. Pessin, J.E., Saltiel, A.R., 2000. Signaling pathways in insulin action: molecular targets of insulin resistance. J. Clin. Invest. 106, 165–169. Rosi, F., Rapetti, L., 2004. Bioactive components in goat milk and plasma. In: Proceedings of the Xth International Symposium on Ruminant Physiology, Copenhagen (Denmark), August 30–September 4, 2004. J. Anim. Feed Sci. (13 Suppl. 1), pp. 701–704. Savino, F., Grassino, E.C., Fissore, M.F., Guidi, C., Liguori, S.A., Silvestro, L., Oggero, R., Miniero, R., 2006. Ghrelin, motilin, insulin concentration in healthy infants in the first months of life: relation to fasting time and anthropometry. Clin. Endocrinol. 65, 158–162. Soriano Guillen, L., Barrios, V., Chowen, J.A., Sanchez, I., Vila, S., Quero, J., Argente, J., 2004. Ghrelin levels from fetal life through early adulthood: relationship with endocrine and metabolic and anthropometric measures. J. Pediatr. 144, 30–35. SAS, 1999. User’s Guide: Statistics Version 7.03. SAS Institute Inc., Cary, NC, USA. Tschop, M., Wawarta, R., Riepl, R.L., Friedrich, S., Bidlingmaier, M., Landgraf, R., Folwaczny, C., 2001. Post-prandial decrease of circulating human ghrelin levels. J. Endocrinol. Invest. 24, RC19–RC21. Vitali, A., Magistrelli, D., Azevedo, J., Bernabucci, U., Ronchi, B., Rosi, F., 2005. Leptin and puberty in goat. In: Proceedings of the ASPA 16th Congress, Turin (Italy), June 28–30, 2005. It. J. Anim. Sci. (4 Suppl. 2), pp. 383–385. Wilson, N.W., Hamburger, R.N., 1988. Allergy to cow’s milk in the first year of life and its prevention. Ann. Allergy 61, 323–327. Xu, R.J., Wang, F., Zhang, S.H., 2000. Postnatal adaptation of the gastrointestinal tract in neonatal pigs: a possible role of milk-borne growth factors. Livest. Prod. Sci. 66, 95–107. Yaida, N., 1929. Ovarian hormone in blood of pregnant women: ovarian hormone in urine of pregnant women: ovarian hormone in milk of pregnant animals. Trans. Jpn. Pathol. Soc. 189, 93–101. Yokota, I., 2003. Roles of leptin and ghrelin during the perinatal period. Clin. Pediatr. Endocrinol. 12, 57–65.