Milk Leptin in Sows and Blood Leptin and Growth of Their Offspring1'2

Milk leptin in sows and blood leptin and growth of their offspring1'2

N. C. Whitley, *34 D. J. OBrien,*5 R. W. Quinn, t6 D. H. Keisler4 E. L. Walker 47 and M. A. Browns

of Maryland Eastern Shore, Department of Agriculture, Princess Anne 21853: tUniversity of Maryland, Department of Animal and Avian Sciences, College Park 20742: University of Missouri, Department of Animal Science, Columbia 65211: and §USDA-ARS Grazinglands Research Laboratory, El Reno. OK 73036

ABSTRACT: Twenty-one mixed-parity (average 2.4 greater in suckling pigs than in delayed suckling pigs, + 0.49) crossbred sows and their offspring were used to averaging 0.69 + 0.08 and 0.54 ± 0.07 ng/mnL, respec- determine whether sow milk leptin at farrowing was re- tively. Although male pigs were heavier (I' < 0.01) at lated to neonatal serum leptiri and pig growth to wean- birth than female pigs (1,507 + 52 vs. 1.381 + 13 g). ing. During farrowing, pigs were randomly allotted to ADC to weaning and weaning weights were similar for suckling (n = 99) or delayed suckling (n = 89) groups, both sexes. averaging 229 + 14 g and 5.829 ± 323 g. re- with delayed suckling pigs placed in a group pen apart spectively, for all pigs; serum leptin concentrations were from the dam before suckling. Both groups had access not affected by sex of the pig. Milk serum leptin was to heat lamps. Colostruin samples were collected no riot associated \,Vitli litter size, parity, pig birth weight, more than 2 ii after farrowing the first pig. Blood sam- ADG to weaning, or weaning weight. Suckling status ples were collected from all pigs approximately 2 h af- did not influence ADC to weaning or weaning weight of ter farrowing was complete; pigs were then ear notched pigs; neonatal pig serum leptin was not related to birth and returned to their dams. Pig B\V was recorded at weight. weaning weight, or ADC to weaning. These 1.2 ± 0.04 d of age and again at weaning. Milk and results indicate that leptin is not directly related to blood serum were collected after centrifugation; leptin early neonatal growth in the pig: however, more in- concentrations were estimated using RIA. Leptin was depth studies are needed to determine possible indirect detected in colostral milk, as expected, and averaged or long-terni effects of early leptin exposure. 46.0 + 1.1 ng/mnL. Pig serum leptin (P < 0.02) was Key words: growth, leptin, milk, pig, relationship, sow

INTRODUCTION

'The authors thank Sylvester "Vie" Cotton and other staff of the Although leptin is secreted from other tissues, it is University of Maryland Easteri, Shore Farin for assistance in con- produced primarily by white adipose tissue. which also ducting the experiments. contains the leptin receptor. In the pig. along with acli- 2 Ment ion of a trade nanie, proprietary product. or specific equip- pose tissue, expression of the lept.in receptor has been ment does not constitute a guarantee or warrant y by the USDA and does not imply approval to the exclusion of other products that may found in the hypothalamus. anterior pituitary gland. be suitable. ovary, uterine body, liver, kidne y. pancreas, adrenal author: ocwhitle©ncat.edu gland. heart, spleen, lung, intestine, bone marrow. and 'Current address: North Carolina AT State University CEP. P0 muscle (Lin et al., 2000). Therefore, it is possible that Box 21928. Greensboro, NC 27120-1928. leptin can influence a variety of physiological functions. 'Current address: Delaware State Universit y. College of Agricul- ture and Related Sciences. Baker Annex Building Room 203. 1200 Leptiri has also been found in the placenta (Hoggard North Dupont Avenue. Dover. DE 19901. et al., 1997; Ashworth et al., 2000). where it passes 'Current address: Children's Merc y Hospitals and Clinics, Build- through to the fetus during its transition td) the neonate ing A, Thomas D. Morris. Inc.. .1001 Millender Mill Rd., Reister- (Matsuda et al., 1999) so both maternal and fetal con- town, MD 211136. centrations of leptin are increased during pregnancy. Current address: Missouri State Universit y, Department of Agri- culture. 207 Karls Hall. Springfield, MO 65897. Leptin is also produced by the mammary gland, anti Received October 15, 2008. has been found in the milk of several species. includ- Accepted January 20. 2009. ing goats (Whitley et al., 2005). sheep (lcFadin et

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1660 \\'hitley et al. al.. 2002), pigs (Estienne et al., 2000). horses (Salimei glass tubes without additive (VWR. Bridgeport, NJ), et al.. 2002), cattle (Wathes et al., 2007), and humans sex of the pig was recorded (40 male. 59 female suck- (Houseknecht et al., 1997). Additionally, in a review of led: 44 male, 45 female urisuckled), and pigs were ear- obesity in humans and early life programming, Cottrell notched for permanent identification. After sampling, and Ozanne (2007) confirmed a small protective effect all pigs were returned to sows, with no cross-fostering of breast-feeding against, later obesity, and suggested conducted. Blood samples were allowed to clot at 4CC that early ingestion of leptin in milk by the neonate overnight and serum was collected after centrifugation might influence later growth and development. Little at 2,500 x q at 4°C for 15 min. Milk samples were research has been conducted to determine the relation stored at —20°C until being ultracentrifuged at 100,000 of leptin in pigs to preweaning growth. Therefore, the x q at 5°C for 1 Ii. The clear supernatant (milk serum) objective of this experiment was to investigate possible was removed and stored at —20°C until analysis. Leptin relationships among sow milk and pig serum leptin and concentrations in blood serum and milk serum were subsequent pig growth to weaning. nieasured using the leptin RIA described by Delavauci et al. (2000) and previously validated for use with milk MATERIALS AND METHODS seruni (McFaclin et al., 2002). Inhibition curves gener- ated by dilutions of pig serum from 25 to 350 1iL were All animal-related procedures were approved by the parallel to the standard curve. University of Maryland Eastern Shore Institutional An- At 1.2 ± 0.04 d of age, pigs were weighed and pro- imal Care and Use Committee. cessed (100 mg of iron dextran injected i.m., needle teeth and tails clipped, males castrated). Pigs were Animals and Procedures weaned at 23.8 + 0.31 d of age. and BW and number weaned were recorded. Average daily gain was cal- Twenty-one mixed-parity (average 2.4 ± 0.49) cross- culated as grams per day of BW gain. Stillbirth and bred sows (PlC x Landrace x Yorkshire) and their preweaning mortality BW were recorded up to the date offspring at the University of Maryland Eastern Shore of pig processing. were used during May to July of the first year. After Al using pooled semen from 2 crossbred boars, sows Statistical Analysis were housed in standard gestation stalls in a comnmer- cial-type, curtain-sided swine facility on a natural light Data were analyzed using mixed model least squares cycle. Sows had ad libitum access to water and were fed procedures (SAS Inst. Inc.. Cary. NC) with P < 0.05 a commercially pelleted. 14% CP gestation ration with considered significant. The initial linear model used in a minimum of 3% crude fat (14% Warm Environment the analyses of individual pig data included the fixed Gestation ration. Southern States Inc.. Gettysburg, PA) effects of parity, treatment. sex, weaning age (linear), at approximately 2.7 kg/d until after farrowing. Sows all possible interactions among fixed classification ef- were moved to environmentally controlled rooms with fects, and the random effects of sow within parity and a 12:12 liglit:dark cycle approximately 7 d before the the pooled random interaction of sow within parity expected date of farrowing for the first animal mated. with fixed effects. Sow was cross-classified with treat- Sows were observed during the farrowing process. and ment and sex, and sow was the main unit experimental a colostrum sample was obtained within 2 It of farrow- unit. Pigs within sow and treat iuent were time subunit ing but after farrowing at least one pig. In the next experimental unit. feeding period after farrowing (within 12 h), sows were Models were reduced in it procedure by switched to a commercially pelleted, 16% CP lactation elimination of unimportant 3-factor fixed interactions feed that was offered on an ad libitum basis (16% Mega (P > 0.25) arid 2-factor fixed interactions, in that or- Sow Lactation, Southern States Inc.). An average of der. In accordance with appropriate model reduction 11.5 + 0.7 pigs were horn live per sow (total litter size procedures, factors (main effects and lesser order inter- of 12.3 ± 0.2. including stillborns): however, only ap- actions) included in significant interactions (P < 0.25) parently health, phenotypically normal pigs that were were left in time linear model independent of their level observed being delivered were used (it 188: 9.0 + 0.7 of significance. Additionally. estimation of subclass pigs/sow). means required inclusion of interactions in the linear During farrowing of each sow, pigs were randomly niodel for estimabilitv. Parity effects were tested by sow allotted to groups of stickling (n = 99) or delayed suck- within parity, and other fixed effects were tested by ling (n 89). Suckling pigs were left with the dam with the pooled error of sow within parity interactions with access to a heat lamp, whereas delayed suckling pigs fixed effects. were placed in a group pen with a heat lamp apart from The relationships of pig serum leptin concentrations the dam in the same room for a maximum of 8 h. Ap- to birth weight. preweaning ADG, and weaning weight proximately 2 h after the end of farrowing (to allow all were analyzed by including serum leptin in the above suckled pigs to ingest colostrum), blood samples were linear mode, with model reductions performed as de- collected from an anterior vena cava from all pigs on scribed previously. The initial linear model used in the the study (1 mL), placed into 12 x 75 mm borosilicate analyses of sow data included the fixed effects of parity,

Sow milk and pig leptin 1661 Table 1. Least squares means (+SEM) for variables measured in male and female pigs allowed to suckle from the dam (suckling) or not suckle from the dam (delayed suckling) until 2 h after farrowing was complete

Pig sex and siickl jug status

Variable Male (it) Female (ii) Delayed suckling (ii) Stickling (11)

Scrum leptiri. ng/inL 0.61 ± 0.08 (77) 0.62 ± 0.07 (89) 1.5.1 ± 11.07" (85) 0.69 ± 0.081 (81)

Birth weight, g 1.507 ± 52 (84) 1,382 ± 43 (104) 1.424 ± 45 (89) 1,466 + 45 (99)

ADC to weaning, S 228 + 15 (59) 230 ± 13 (68) 238 13 (62) 220 ± 13 (65)

Weaning weight, g 5.872 + 343 (59) 5.785 + 303 (68) 5.991 ± 303 (62) 5.667 ± 305 (65) ""Within suckling and row. iiieans without a cmnioii superscript differ (P < 0.02). "Withinhiis sex and row, means vi (hunt a common superscript differ (P < 0.01). treatment. parity x treatment, and weaning age (lin- stomach in rat pups. and Berg et al. (2007) noted that ear) and the random effects of sow within parity and lel.)tin concentrations in neonatal foals increased sig- sow x treatment within parity. Model reductions were nificantly after nursing. In contrast, goat kids removed performed sinularl y to the procedure given for the anal- from the dam at birth and bottle-fed colostrum within yses of individual pig data. The relationships of sow 3 h after birth did not have greater concentrations of milk leptin concentrations to birth weight. preweaning serum leptin at 6 Ii after feeding than before colostrum ADG, and weaning weight were analyzed by including feeding (Rasmussen et al., 2008). Therefore, although milk leptin in the above linear model as a. covariate, leptin certainly has the potential to play a role in the With model reductions performed as described above. development of the neonate either systemically or lo- cally at the level of the gastrointestinal tract, more in- RESULTS AND DISCUSSION tensive studies are needed. Pig serum lept.in was not related to birth weight in this In the present study. leptin was found in the colostral study. Because neonatal pigs have decreased amounts of milk serum of sows (averaging 46.0 ± 1.1 ng/mL), and adipose tissue in general and lack brown adipose tissue pig serum leptin was greater (P < 0.02) for suckling (Herpimi et al., 2002). the well-documented positive re- than for delayed suckling pigs (Table 1). Colostral milk lationship between body fat (especially brown adipose was reported to have increased concentrations of leptin tissue) and seruni leptin concentrations might explain in other studies with pigs (Estienne et al., 2000) as this result. In agreement with the present study, lamb well as for other species, including meat goats (Whit- birth weight was independent of body fat and was thus ley et al.. 2005). sheep (McFadin et al., 2002), and independent of plasma leptin concentrations (Ehrhardt mares (Saliniei et al.. 2002; Berg et al., 2007). The in- et. al., 2003). In humans, a species with adequate neo- creased concentrations of leptin in periparturient milk natal adipose tissue, cord serum leptin at birth was was theorized to he attributed to a pooling of leptin in positively correlated with birth weight (Tamura et al. the udder before parturition in sheep (McFadin et al., 1998; Perrone et al., 2000: Martinez-Cordero et al.. 2002), although Rasmussen et al. (2008) indicated that 2006) but was not independent, of percentage of body prepa.rtum accumulation of leptin in colostrum did not fat (Martinez-Cordero et al., 2006). Pig serum leptin seem to occur in dairy goats (it 4). at birth was also not significantly related to weaning Overall, it is accepted that relatively increased con- weight or ADC in the present study. Because pigs with centrations of milk leptin occur at parturition, which delayed suckling were not offered liquids during sepa- coincides with the time when neonates are best able ration and were placed hack on their danis within 8 to absorb large proteins through the gastrointestinal h of removal, ingestion of some colostrum (amid thus tract, and evidence does exist that the neonate can some leptin) could still have occurred. In other species. absorb leptin ingested orally. For example, as with the colostral leptin decreased quite dramatically by 12 Ii present study, elevated blood serum leptin was found after parturition (McFadimm et. al.. 2002; Whitley et al., in neonatal suckled compared with unsucklecl rat pups 2005; Berg et al. 2007), which could point to relatively (Dessolin et al.. 1997). Blood leptii was greater in rat negligible concentrations being ingested by the pig in pimps fed milk plus leptin vs. milk alone (Casabiell et the delayed suckling group if the same were true for al.. 1997). in neonatal pigs suckled vs. being fed milk sows, but Milk serum lept.in has not been measured in replacer (Weiler et al., 2002), in neonatal calves fed frequent intervals after parturition in this species. How- colostrum vs. milk replacer (Blum et al., 2005), and in ever, because suckling status did! not influence ADG to breast-fed compared with formula-fed human infants weaning or weaning weight of pigs (Table 1), the de- (Savino et al., 2004). In addition, Sanchez et al. (2005) layed ingestion of leptin would likely not have affected noted that leptin is absorbed through the neonatal the results of the study.

1662 Whitley et al. Milk serum leptin was not associated with litter size, Cottrell. E. C.. and S. E. Ozanne. 2007. Early life programming of pig birth weight, ADC; to weaning, or weaning weight. obesity and metabolic disease. Pliysiol. Behav. 94:17 28. Breast milk leptin was also not related to infant body Delavaud, C., F. Bocquier, Y. Chilliard, D. H. Keisler, A. Certler. and G. Kann. 2000. Plasma leptin determination in ruminants: mass in humans (Uysal et al.. 2002), and in goats, milk Effect of nutritional status and body fatness on plasma leptin serum leptin throughout lactation was not associated concentration assessed by a specific RIA in sheep J. Endo- with kid BW when measured from birth to 21 d of age crinol. 165:519-526. or from 7 to 56 d of age (Whitley et al.. 2005). Dessolin, S.. M. Sclialling. 0. Champigny, F. Lonnqvist, C. Ailhaud, Suckling status did not influence the average number C. Dani. and D. Ricquier. 1997. Leptin gene is expressed in rat brown adipose tissue at birth. FASEB J. 11:382-387. of pigs weaned per sow (8.6 ± 0.2). The unusually in- Ehrhardt, R. A.. P. L. Greenwood, A. W. Bell,. and Y. H. Boisclair. creased mortality rates were primarily due to crushing, 2003. Plasnia leptin is regulated predominantly by nutrition in which was thought to be caused by increased agitation prerunrinant lambs. J. Nutr. 133:4196-4201. of sows resulting from the novel human interactions Estienne,M.,J.. A. F. Harper, C. H. Barb, and Al. .1. Azuii. 2000. required for the study and the use of several first-parity Concentrations of leptin in serum and milk collected from lactating sows differing in body condition. Domest. Animim. Endo- sows (gilts). crinol. 19:275-280. Although male pigs were heavier (P < 0.01) at birth Herpin, P., D. Damon. and J. LeDividich. 2002. Development of than female pigs, ADG to weaning and weaning weights thermnoregulation and neonatal survival in pigs. Livest. Prod. were similar for both sexes, and sex was not associated Sci. 78:25-45. with serum leptin concentrations (Table 1). There was Hoggard. N., L. Hunter, J. S. Duncan. L. M. Williams. P. Trayhurn. and J. C. Mercer. 1997. Leptin arid leptin receptor mRNA and also no difference in cord blood leptin concentrations protein expression in the murine fetus and placenta. Proc. between male and female limrian neonates (Okereke et Natl. Acad. Sci. USA 94:11073 -11078. al.. 2002), and there was no influence of sex (castrated Housekneclmt. K. L., M. C. McCuire, C. P. Portocarrero, Al. A. males vs. females) on circulating leptin concentrations McGuire, and K. Beermnn. 1997. Leptin is present in human in lambs, although seruni leptin and BW were mea- milk and is related to maternal plasma leptin concentration and adiposity. Biochemn. Biophys. Res. Connnun. 240:742-747. sured over a 47-d period after birth (McFadin et al.. Lin. J., C. R. Barb, H. L. Matteri, R. R. Kraeling. X. Chen, R. J. 2002) compared with only at birth for the pig and hu- Meinersmann. and C. B. Rampacek. 2000. Long form leptin man studies. receptor mRNA expression in the brain, pituitary, and other In the current study, no apparent relationship be- tissues in the pig. Domest. Animn. Endocrinol. 19:53-61. tween darn milk leptin and offspring growth was not- Martinez-Cordero, C., N. Anmaclor-Licona, J. M. Cuizar-Mecloza. J. Hernandez-Medex, and C. Ruelas-Orozco. 2006. Bod y fat at ed. In addition, regardless of suckling status, no rela- birth and cord blood levels of insulin, adiponectin, leptin and tionship was noted for pig serum leptin and growth insulin-like growth factor-I in small-for-gestational-age infants. to weaning. Therefore, as seen in lambs (McFadin et Arch. Med. Res. 37:490-494. al., 2002) and goats (Whitley et al., 2005), the present Matsuda, J., 1. Yokota, M. Idia. T. Murakarni, M. Yamada, T. Saijo, data are consistent with the current lack of convincing E. Naito, M. Ito, K. Shinia, and Y. Kuroda. 1999. Dynamic changes in serum leptin concentrations during fetal and neona- evidence for leptin as a direct modulator of neonatal tal periods. Pediatr. Res. 45:71-75. growth. However, the long-term effects of milk leptin McFadin, E. L.. C. D. Morrison, P. R. Buff, N. C. Whitley, and D. II. and the possible indirect effects of milk leptin on neo- Keisler. 2002. Leptin concentrations in penipartunient ewes and natal development, survival, or other factors not mea- their subsequent offspring. J. Anim. Sci. 80:738-743. sured in this study, such as those theorized for neonatal Mist,rv. A. Si., A. Swick. and D. R. Romsos. 1999. Leptiu alters metabolic rates before acquisition of its anorectic effect in (Ic- gut development (Wolinski et al., 2003), or on thermo- veloping neonatal mice. Am. J. Physiol. 277:R742- R747. regulation (Stehling et al., 1997, Mistr y et al., 1999, Mostyn, A., J. Bispham, S. Pearce, Y. Evens, N. Ravier. D. H. 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