Original Paper Biol Neonate 1992;61:110—117

Agriculture Canada, Lennoxville Research Station, Lennoxville, Canada; University of Montreal, Laboratory of Neuroendocrinology, Notre-Dame Hospital, Montreal, Canada

Key Words Somatostatin immunization Growth hormone-releasing factor Carcass composition Fasting Neonatal swine

Carcass Composition and Resistance to Fasting in Neonatal Piglets Bom of Sows Immunized against Somatostatin and/or Receiving Growth Hormone-Releasing Factor Injections during Gestation

Abstract Thirty-eight gestating sows were either immunized against somatostatin (SRIF) and/or injected with growth hormone­ releasing factor (GRF). Treatment effects on carcass composi­ tion and resistance of newborn piglets to a 60-hour fast were investigated. Protein content of carcasses at birth was in­ creased in piglets of sows receiving GRF or immunized against SRIF, however, when sows received both treatments there was a reduction in carcass protein content (p = 0.01). Other carcass components were unaltered by treatments, and none of the treatments affected metabolic or endocrine pro­ files of piglets at birth. Concentrations of GH, IGF-I (p < 0.01), glucagon and cortisol (p < 0.05) increased linearly with duration of fast, whereas glucose values decreased. Resistance to fasting was unaltered in piglets from any treatment thereby suggesting that exogenous GRF and/or SRIF immunization of sows during gestation are unlikely to improve survival of new­ born piglets.

Introduction Newborn piglets have small birth weights, low body fat and poor energy stores at birth, thereby rendering them particularly suscepti­

ble to death. Studies using diabetic sows have indicated that altering the dam’s plasma hor­ mone and metabolite concentrations could have positive repercussions on the fetuses. Elevated sow blood glucose resulted in signifi-

C. Farmer, PhD Agriculture Canada Lennoxville Research Station P.O. Box 90 Lennoxville, Quebec JIM IZ3 (Canada)

© 1992 S. Karger AG. Basel 0006-3126/92/ 0612-0110S2.75/0

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C. Farmera D. Petitclerca G. Pelletiera P. Gaudreaub P. BrazeaiG

Materials and Methods Animals and Treatments Second parity Yorkshire X Landrace sows were used in a 2 X 2 factorial experiment. Animals were immunized against SRIF (SRIF-IMM) and/or re­ ceived GRF during gestation. The treatments were: (1) saline injections and immunization against bovine serum albumin (BSA), n = 11; (2) saline injections and SRIF-IMM. n= 10: (3) GRF injections and BSA-IMM. n = 9, and (4) GRF injections and SRIF-IMM, n = 8. Immunizations against SRIF or its conjugated protein (BSA) were done at 30, 44, 58, 72, 86 and 100 days of gestation while GRF injections (3 ml, 9 mg/injection) were given subcutaneously at 08.00,14.00 and 20.00 h daily from day 90 of gestation until parturition. Linear SRIF-14 was coupled to BSA (IAF Biochcm. Laval. Canada) in a ratio of 1:1.3 (w/w) with 68% efficiency. For the first immunization, the antigen (BSA-SRIF) dissolved in saline was mixed with Freund’s complete adjuvant in a ratio of 1:1 (v/v). A total of 3 ml of this emulsion containing 1.5 mg of SRIF was injected intradermally (20 sites; 2 ml) in the neck area and subcu­ taneously (4 sites: 1 ml). In subsequent immuniza­ tions, 3 ml of the emulsion (containing 1 mg of SRIF) with Freund’s incomplete adjuvant were injected sub­ cutaneously (10 sites). Control animals received BSA in a similar manner. During gestation, sows were fed 2.5 kg/day of con­ centrate (14.2% crude protein) and 0.3 g/day of wheat bran as top dressing. Sows were transferred to raised farrowing crates on day 80 of gestation, and farrowing was induced with prostaglandin on day 113 (Planate®, Coopers Agropharm, Willowdale, Canada; 2 ml i.m., 184 pg). All piglets were weighed and sexed at birth and, unless they weighed less than 0.8 kg. piglets from birth order number 2-5 were placed in individual cages in a controlled atmosphere room. Temperature was kept at 31 ± 1 °C, and piglets were fasted for 60 h while hav­ ing free access to sterile water. Water was provided in rabbit drinking bowls with inverted water bottles in them. Blood samples (6 ml) were obtained from the suborbital sinus of all fasted piglets at 0, 20, 40 and 60 h after farrowing as well as rectal temperature and body weight. A blood sample was also obtained at birth from the suborbital sinus of piglets number 1,6. 7 and 8 in the birth order. These last piglets were then sacri­ ficed by exsanguination after stunning, they were evis­ cerated, and the liver was promptly removed, weighed and frozen in liquid nitrogen, after which it was kept on dry ice until stored at -8 7 °C . The head, tail, hooves and viscera were removed from the carcasses

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cantly increased fetal glucose [1-3] and free fatty acid (FFA) concentrations [1, 3] as well as increased percent body fat [1, 2, 4] and liver glycogen [4] at birth. Exogenous GH can also be used to manipulate circulating levels of maternal metabolites and hormones. Injec­ tion of sows with porcine GH during the last 14 or 21 days of gestation resulted in a diabe­ togenic state [5, 6], The 21-day injection pe­ riod increased total body lipids in newborn piglets and had a tendency to increase FFA concentrations of piglets at birth [6], A trend to greater glucose concentrations after a 24hour fast was observed in piglets from treated sows in both studies [5, 6]. Survival of the pig­ lets to 21 days postpartum was not affected by sow treatment [6], but the authors postulated that a more immediate test of survival, such as a 60-hour fast, may be necessary to deter­ mine the advantage of improved energy ho­ meostasis during the critical first 3 days after birth. In fact, when piglets from diabetic gilts were subjected to a 60-hour fast, they showed greater survival than control neonates [3]. Active immunization against somatostatin (SRIF) in gestating gilts may also prove bene­ ficial to piglets, since newborn pigs from im­ munized sows had greater circulating levels of glucose and FFA at birth than control piglets [71Elevated plasma glucose concentrations and faster cumulative weight gain were also seen in lambs born of ewes immunized against somatostatin [8], The present experi­ ment was therefore carried out to determine the effects of exogenous growth hormone­ releasing factor (GRF) and/or active immuni­ zation against somatostatin during gestation of sows on carcass composition of piglets at birth and on resistance to a 60-hour fast of newborn piglets. Hormonal and metabolic re­ sponses to fasting were also determined.

Peptides Porcine GRF (1-29) NH; was provided by Sanofi Recherche, Montpellier, France. SRIF was synthesized by solid phase methodology [9] and purified (Neuroen­ docrinology Laboratory, Notre-Dame Hospital, Mon­ tréal, Canada) by high pressure liquid chromatography [10]. Purity of GRF was 96-97% with a peptide con­ tent of 83-86%. Purity of SRIF was 98% with a pep­ tide content of 93 %. Stock solutions of GRF were pre­ pared at concentrations of 5 mg/ml in 520 pi of 0.1 N HC1; volume was then adjusted by adding 480 pi of 0.1 N NaOH. Subsequent dilutions were prepared in 0.9% NaCl. Hormone and Metabolite Assays Porcine GH was assayed by an established ra­ dioimmunoassay (R1A) [11], The first antibody (Dr. G. DesCotes, Sanofi Recherche) showed no cross-reac­ tivity with 1 pg m H of thyrotropin, luteinizing hor­ mone, follicle-stimulating hormone or 5 pg ml-1 of PRL. The intra- and interassay coefficients of varia­ tion were 9.1 and 9.4%, respectively. Sensitivity of the assay was 0.2 ng ml-1. Concentrations of IGF-I were determined according to a previously described RIA [7, 12]. The first antibody was graciously donated by Drs. L. Underwood and J.J. Van Wyk and the Na­ tional Institute of Diabetes and Digestive and Kidney Diseases through the National Hormone and Pituitary Programme, University of Maryland School of Medi­ cine. Sensitivity of the assay was 6 pg/tube and intraand interassay coefficients of variation were 9.3 and 11.8 %, respectively. Porcine insulin concentrations were determined according to a double-antibody assay procedure [12]. Sensitivity of the assay was 20 pg/tube and intra- and interassay coefficients of variation were 6.0 and 14.0%, respectively. Serum concentrations of PRL were quantified by a homologous double­ antibody RIA procedure [13]. The sensitivity of the assay was 0.1 ng/tube. The intra- and interassay coeffi­ cients of variation were 11.8 and 13.3 %, respectively. Cortisol concentrations were determined according to

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a validated RIA procedure [12]. The specificity of the antibody was tested against 14 other steroids and was less than 0.1 % at 1:3,000 dilution, except for 11-deoxycortisol (6%), corticosterone (0.5%), prednisolone (0.5%) and 17-a-OH-progesterone (0.2%). The sensi­ tivity of the assay was 10 pg/tube. The intra- and interassay coefficients of variation were 8.0 and 18.4%, respectively. Blood samples for glucagon assay were collected in aprotinin (500 kallikrein inactivator units/ml; Sigma, St. Louis, Mo., USA) and plasma was harvested. For the assay, 200 pi of assay buffer (0.02 M glycine. 1% BSA; 0.01% Merthiolate, pH 8.8), 200 pi of first antibody (04A beef-pork glucagon antibody provided by Dr. R.H. Unger, University of Texas, and with specificity similar to Unger’s 30 kD antibody; 1:225,000 final antibody dilution) and 200 pi of standard (2.5, 5.0, 10, 20. 40. 80, 160 and 320 pg of glucagon injectable, USP; Eli Lilly, Canada) or plasma were incubated for 24 h at 4°C. Then, 100 pi of iodinated glucagon (8,000 cpm; [3-(l25I)iodotyrosyl10] glucagon; IM-160, Amersham, Oak­ ville, Canada) was added, mixed and incubated for an additional 48 h. After this period, 1 ml of second anti­ body (1:166 dilution in assay buffer) containing PEG (42.75 g/1) was added and incubated at 4°C for 1 h: then, 100 pi of normal rabbit serum (1 %) was added and incubated for an additional 30 min. Tubes were subsequently centrifuged at 2,000# for 20 min, super­ natant was decanted, tubes were dried and counted in a y-counter. Eighty percent binding was 3.9 pg/tube; intra- and interassay coefficients of variation were 4.6 and 8.9%, respectively. Serum glucose (Boehringer Mannheim, Meylan, France) and FFA (Boehringer Mannheim, Tovanvmon, Min Ato-Ky, Tokyo, Japan) concentrations were determined using an automatic Hitachi No. 705 blood analyzer. Daily calibration of the blood analyzer was made using the Wellcome Clinical Chemistry Quality Control Program (Wellcome Foundation, Temple Hill. Dar.ford, UK). Specific binding of l25I-labelled Tyr'-SRIF by serum (1:150 dilution) was measured using a previously described RIA [7], Nonspecific binding was less than 5%. If serum from sows showed less than 5% binding capacity on day 72 of gestation, these animals were removed form the study. Chemical Analyses Carcasses w'ere ground three times in a meat grinder through a plate with holes 5 mm in diameter. They were then put in a blender (15 A) until homoge­ nous in texture. The samples were weighed, freezedried and weighed again, to determine moisture con­ tent. Before performing the chemical analyses, the

Farmcr/Pctitclcrc/Pelletier/Gaudrcau/ Brazeau

Fasting in Neonatal Swine

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which were then frozen at -2 0 °C until analyzed for fat and protein content. Blood samples were either put on ice and centri­ fuged (2,000#, 15 min) within 2h (for glucose and FFA analyses) or left at room temperature for 4 h, stored overnight at 4°C and centrifuged (2,000#, 15 min) the following day to yield serum for antibody titer and hormonal assays. For glucagon assay, plasma was prepared within 20 min of blood collection, using EDTA as anticoagulant and 500 kallikrein inactivator units of aprotinin/ml of blood.

Table 1. Carcass composition, hormonal and metabolic values of newborn piglets before initial suckling Carcass composition body dry fat weight, kg matter, % %

Mean SEM n1

1.23 0.01 380

79.1 0.1 132

1.2 0.01 132

Blood variables liver glycogen, %

FFA ]iEq/l

glucose mA//l

GH ng/ml

IGF-I ng/ml

insulin ng/ml

PRL ng/ml

cortisol ng/ml

11.9 0.2 132

52.9 1.4 360

3.28 ' 0.07 360

95.4 3.3 361

34.7 0.6 361

10.5 0.3 347

6.7 0.2 292

218.2 5.1 361

freeze-dried preparations were pulverized in a cen­ trifugal grinding mill (Brinkmann ZM-1; Brinkmann Instruments, Rexdale, Canada). Protein was deter­ mined by micro-Kjeldahl (Kjeltex Auto System; Tecator AB, Hoganas, Sweden) and fat was extracted twice with dichloromethane using the Soxtex System (Tecator 1043 Extraction Unit, Tecator, Herudan, Va„ USA). Crude fat was determined by weighing the extracted residue. Liver glycogen was determined us­ ing the anthrone reagent following cold trichloroacetic acid extraction as described by Randall and L’Ecuyer [14]. Statistical A nalyses The General Linear Model Procedure of the SAS Institute [15] was used for statistical analyses. Data from piglets at birth were analyzed according to a split plot design with treatment, sow within treatment and sex as main factors. Sow within treatment was the error term for treatment and sow (treatment) X sex was the error term of sex and treatment X sex. Treat­ ment effects were determined with orthogonal con­ trasts according to the factorial arrangement. Data from fasting piglets were analyzed as a split split plot with hour added as a main effect. Orthogonal contrasts were then run on the variable hour. The error terms used to test the contrasts of repeated measurements were partitioned according to Rowell and Walters [16].

Results All SRIF-IMM sows used in the trial had > 5 % serum binding to SRIF (1:150 dilu­ tion). There was a significant GRF X SRIF-

IMM interaction (p = 0.01) on carcass protein content of piglets at birth (controls = 11.5%, GRF = 11.8%, SRIF-IMM = 12.1 %, GRF X SRIF-IMM = 11.3%; SEM = 0.2). There was no significant effect of treatments on other carcass composition variables, body weight, glycogen reserves or any of the blood vari­ ables measured at birth. Average values across treatments are presented in table 1. Male piglets, on the other hand, were heavier (p < 0.01) at birth than female piglets (X ± SEM; 1.26 ± 0.02 vs. 1.19 ± 0.02 kg)L_had higher (p < 0.01) carcass dry matter (X ± SEM; 79.6 ± 0 . 1 5 vs. 78.8 ± 041%) and lower (p < 0.01) carcass protein (X ± SEM; 11.4 ± 0.11 vs 11.9 ± 0.09%). There was also a tendency (p = 0.075) for males to have greater liver glycogen than females at birth. However, the GRF X SRIF-IMM by sex interaction was significant for glycogen (p = 0.055) and the GRF by sex interaction was significant for dry matter (p < 0.01) content (table 2). Sex had no effect (p > 0.05) on hor­ monal values of piglets at birth and none of the interactions were significant. Data from fasting piglets showed that over­ all, PRL concentrations were lower in piglets from SRIF-IMM sows compared to piglets from BSA-IMM sows (table 3; p < 0.05); there was no interaction with hour. There was also a tendency (p = 0.09) for GRF X SRIF-

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1 Number of observations.

Table 2. Liver glycogen and carcass dry matter (DM) in male (M) and female (F) newborn piglets of sows injected with GRF and/or immunized against SR1F during gestation GRF

Control

Liver glycogen, % Carcass DM, %

SRIF-IMM

GRF-SRIF

M

F

M

F

M

F

M

F

12.2 80.1

11.4 78.8

12.7 79.2

11.6 78.8

12.2 79.5

11.1 78.3

12.5 79.2

12.4 79.6

SEM

0.41 0.22

Table 3. PRL concentrations (ng/ml; X) during fasting in piglets from sows receiving GRF and/or immunized against SRIF during gestation Hour

Control

GRF

SRIF-IMM

GRF X SRIF-IMM SEM

0 20 40 60

8.4 3.2 6.0 5.2

7.1 4.3 5.4 4.5

6.0 3.3 4.3 4.3

5.5 4.2 5.2 5.0

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(6.8 °C decrease). Concentrations of GH, IGF-1 (p < 0.01), glucagon and cortisol (p < 0.05) increased linearly with duration of fast, whereas glucose values decreased linearly (p < 0.01) and quadratically (p < 0.05; table 4).

Discussion Immunization of sows against SRIF and/or injections of GRF during gestation had no beneficial effects on the energy status of piglets at birth or on their resistance to fast­ ing. Sows in the GRF and SRIF-IMM groups deposited more protein in their fetuses than control and GRF X SRIF-IMM sows, yet birth weight was unaffected. There is no ex­ planation for this finding. Even though the decrease in rectal temperature was lesser in fasted piglets from SRIF-IMM sows, this was not translated into a greater survival rate. The lack of effect on survival of piglets can be

Farmer/Petitclerc/Pelletier/Gaudreau/ Brazeau

Fasting in Neonatal Swine

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IMM piglets to have decreased cortisol values in comparison to controls (30.0 ± 0.2 vs. 34.9 ± 0.3 ng/ml). No other variables, including survival of piglets, were affected by any of the treatments (p > 0.1). The average percent survival of piglets among treatments ( ± SEM) for 20, 40 and 60 h of fasting were 99.3 ± 0.7, 97.9 ± 1.2 and 74.3 ± 4.9%, respectively. Male piglets were heavier (1.3 and 1.1 kg at 0 and 60 h vs. 1.2 and 1.0 kg for females, SEM = 0.02; p < 0.01) and had higher insulin concentrations (10.6 and 10.6 ng/ml at 0 and 60 h vs. 10.6 and 8.8 ng/ml for females, SEM = 1.3; p < 0.05) than females throughout the fast. Duration of the fast sig­ nificantly altered all of the measured vari­ ables except for insulin and PRL (tables 3 and 4). Body weights and rectal temperature de­ creased linearly (p < 0.01) with time and the slope of the decrease in rectal temperature was lower (p < 0.05) for piglets from sows immunized against SR1F (5.4 °C decrease) compared to piglets from BSA-IMM sows

0.4 0.2 0.3 0.3

Table 4. Weight, temperature, hormonal and metabolic values of newborn piglets during a 60-hour fast (X ± SEM)1 Hour

Body weight kg

Rectal temperature °C

FFA HEq/1

Glucose m M fi

GH ng/ml

IGF-I ng/ml

Insulin ng/ml

Glucagon pg/ml

Cortisol ng/ml

0

1.27 ±0.02 (140)

99.8 ±0.2 (136)

51.4 ± 1.9 (140)

3.2 ±0.1 (140)

88.8 ±4.6 (140)

34.7 ±0.9 (140)

10.6 ±0.5 (134)

208.2 ±14.9 (138)

212.4 ±7.4 (140)

20

1.19 ±0.02 (135)

98.7 ±0.1 (127)

74.4 ±2.3 (139)

3.8 ±0.1 (139)

197.8 ±7.1 (139)

86.8 ±3.9 (139)

10.1 ±0.7 (116)

124.2 ± 12.5 (137)

164.3 ± 10.2 (139)

40

1.12 ±0.02 (129)

96.5 ±0.2 (137)

36.8 ±2.1 (137)

2.5 ±0.1 (137)

306.9 ±7.6 (137)

155.5 ±5.7 (137)

10.4 ±0.4 (121)

360.6 ±24.1 (132)

313.3 ± 18.9 (136)

60

1.07 ±0.02 (102)

93.7 ±0.2 (104)

45.8 ± 1.9 (104)

1.3 ±0.1 (104)

330.7 ±10.6 (103)

183.8 ±6.3 (105)

9.8 ±0.5 (93)

501.1 ±27.0 (104)

609.8 ±34.0 (103)

p valuesHour linear Hour quadratic Hour cubic Interactions

or receiving growth hormone-releasing factor injections during gestation.

Thirty-eight gestating sows were either immunized against somatostatin (SRIF) and/or injected with growth hormone-releasing factor (GRF). Treatment ef...
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