Body composition and blood parameters of newborn piglets from Alentejano and conventional ( Large White × Landrace ) genotype

This study aimed to compare the body composition and some blood parameters [(glucose, albumin and insulin-like growth factor 1 (IGF-1)] of newborn piglets farrowed by unselected Alentejano (AL) or conventional genotype [LargeWhite Landrace (LL)]. Carcasses (12 of each genotype) and blood samples were obtained from a total of 34 litters (18 AL, 16 LL). Gestation length was 4d shorter in AL sows compared with LL sows. When adjusted for birth weight, carcasses of AL piglets showed higher percentages of dry matter (P < 0.05) and crude protein (P < 0.01) and tended to have higher lipid content (P = 0.091) than carcasses of LL piglets. Relative to body weight, the AL piglets had heavier livers (P < 0.05) than LL piglets but glycogen content was similar in both genotypes. Longissimus dorsi muscle of AL piglets contained more protein (P < 0.01), but glycogen, DNA and RNA contents were similar in both genotypes. The blood from the AL piglets had higher levels of glucose (P < 0.01), albumin (when adjusted for birth weight) (P < 0.05) and IGF-1 (P < 0.05) than blood from the LL piglets. On the bases of body composition and studied blood parameters, AL piglets seem to be more mature at birth than LL piglets despite a shorter gestation lenght. Additional key words: body protein, energy stores, Iberian neonatal pig, maturity, plasma parameters.


Introduction
Alentejano swine (AL) is a primitive Iberian breed (IB) raised all over the Southwest region of the Iberian Peninsula.The AL breed is well-adapted to utilise acorns and is raised mainly in an extensive system, under oak canopy (green and cork) in Alentejo region (Portugal).In most farms, sows are raised in outdoor parks.Due to the seasonal and annual variations in natural resources in the Mediterranean Quercus woodland ecosystem (acorns production from October to March, pasture in autumn and spring), they are usually supplemented with standard commercial feeds.
Iberian breeds have a remarkable ability to deposit fat as intramuscular and external fat (López-Bote, 1998) but a reduced potential for protein deposition (Nieto et al., 2002).Nevertheless, they have an important commercial value in the local economy through their long dried cured hams and sausages.However it is not known whether their ability to deposit fat is already expressed at birth.Further, compared to conventional breeds (e.g., Large White), reproductive performance of the IB breeds is quite low.Litter size ranges from 7.4 to 8.4 total born (Dobao et al., 1988;Marques, 2001) while pre-weaning mortality of piglets can be as high as 28-29% (Marques et al., 1996;Robledo et al., 2008).Iberian sows have a gestation length by approximately 4d shorter than conventional sows (De Juana Sardón, 1954;Nunes, 1993).In pigs, growth rate of the foetuses (McPherson et al., 2004), and vital deposition of energy reserves (Okai et al., 1978) accelerate markedly during the last few days of gestation.However, to what extent these are affected by the shorter gestation length of the IB sows has not been determined.
Less is known on the early stages of development of the IB pig.Usually, premature birth (4-5d before the normal term) reduces birth weight of piglets and enhances post-natal mortality (Yamada et al., 1982).Compared to the genetically obese Ossabaw (Stone et al., 1985) or Meishan piglets (Herpin et al., 1993), newborns from lines selected for lean tissue growth have less dry matter and protein concentrations in their body and lower liver weight.Further, 21 years of genetic selection of the French Large-White (LW) on lean growth and litter size also resulted in similar effects on the newborns (Canario et al., 2007b).
This study aims to compare the body composition and some blood parameters of newborn piglets farrowed by the unselected AL, reared as in practice, or by a more conventional [LW × Landrace (LR)] genotype sow.

Animals and facilities
The experiment was conducted according to the European Community regulations concerning the production of experimental animals (European Communities Council Directive 86/609/EEC).The experiment was carried out in the Experimental Centre of Mitra of the University of Évora (Portugal) from February 2005 to August 2006.The AL sows were mated by AL boars whereas Large-White × Landrace (LL) sows were mated by a LW boar.Sow parity number ranged from 3.7 ± 0.6 to 4.0 ± 0.6 for AL and LL sows, respectively.Litters were followed until 28d post-partum.
During the whole gestation, LL sows were kept in groups of 3 to 4 sows, as homogeneous as possible in live-weight and age, in indoor facilities on concrete flooring.To control mating and to determine the duration of gestation, AL sows were kept in group of 3 to 4 sows in indoor facilities until gestation was confirmed by ultrasonic (Aloka; Model: SSD-210DX II, with a 5 Mhz linear probe) diagnosis (~ d 28 after mating).Thereafter, they were moved to and raised in groups of 12-15 sows in an outdoor park (4.5 ha) where they had access to floorless huts.Alentejano sows farrowed in conventional farrowing crates (indoor farrowing house) due to experimental purposes (piglets slaughter and bleeding, colostrum and milk samples collection).Sows were fed with commercial diets for gestating and lactating sows.Diets were based on cereals and soybean meal and contained (per kg diet, calculated values) 160 g crude protein (CP), 7 g lysine and 13.4 MJ digestible energy (DE) (gestation) and 170 g CP, 10.5 g lysine and 13.8 MJ DE (lactation).During gestation, LL sows were fed twice daily at the rate of 2.5-3.0 kg d -1 until farrowing.Alentejano sows were fed with the same commercial diets as LL sows, at the exception that the rate of feeding was restricted to 1.5-2 kg d -1 during gestation.However, for 6 out of the 18 AL sows which had access to acorns, feed supply was restricted to 1 kg d -1 .For both genotypes no feed was provided during the farrowing day, after which feed supply was gradually increased until ad libitum.In the outdoor park they were fed once daily the same type and amount of feed.Feed was spread in the field in order to reduce compe-tition between sows.At mating and one day before the expected farrowing date, AL and LL sows weighed 127 ± 12 kg and 161 ± 14 kg, and 173 ± 9 kg and 227 ± 11 kg, respectively.Due to the fact that sows were kept in groups, individual feed intake was not determined.In all facilities, water was available from a low-pressure nipple-drinker.

Measurements
A total of 34 litters (18 AL, 16 LL) were used for the determination of body composition and blood sampling at birth.Sows farrowed naturally and all farrowings were supervised.On the day after farrowing the backfat thickness (BFT) was determined on P2 site (Dourmad et al., 2001).The body composition of piglets at birth was determined on 12 AL and 12 LL piglets (one piglet per litter).Of the 12 AL piglets, six were farrowed by AL sows which had access to acorns during gestation.At birth, randomly chosen piglets were dried, weighed and exsanguinated.Duration of these operations did not exceed 3 to 4 min.The digestive tract, lungs, heart and liver were removed and the liver (without gall bladder) was weighed.Longissimus dorsi muscle (LD) samples (5-6 g) were taken.The liver and LD samples were frozen in liquid N 2 and stored at -20°C until analysed.Piglet carcasses without the digestive tract, liver, lungs and heart were also frozen and stored at -20°C until subsequent mincing and homogenization.Blood samples (~1.0-1.5 mL) were collected into heparinised tubes after umbilical cutting for the determination of glucose, albumin and insulin-like growth factor 1 (IGF-1).Samples were immediately centrifuged at 1,400 g during 10 min and the plasma removed and stored at -20°C until analysed.Glucose was determined on 63 AL piglets from 15 litters and on 91 LL piglets from 14 litters.Albumin was determined on 89 piglets (45 AL and 44 LL piglets from 13 and 10 litters, respectively).Piglets were chosen so that within each genotype they had the same gestational age, i.e., 111 ± 1 and 115 ± 1d for AL and LL piglets, respectively.Within a litter, piglets having a birth body weight higher than litter average birth body weight were considered heavy and the others were considered light.For IGF-1, samples were from 3 piglets per litter (5 AL and 6 LL litters) chosen as being heavy, medium (a medium piglet had the closest birth body weight to the litter mean birth weight) or light within litter.

Analyses
Dry matter (DM), CP, lipid, fatty acids and ash content of the ground carcasses were determined.The DM was determined after drying at 102°C until constant weight.The CP (N × 6.25) was determined according to Dumas method using a LECO (Ref.FP-528, LECO Corporation, St Joseph MI, USA) nitrogen/protein determinator.Total lipid content was measured according to Folch et al. (1957) and methyl esters were obtained.Fatty acids were determined by gas phase chromatography (Chromatographer: GC-FID HP 6809 Series).The ash content was determined after incineration at 550°C for 3 h.Glycogen concentration of liver and LD was measured as previously described by Le Dividich et al. (1991).Glucose-6-phosphatase activity (G6-Pase), a key gluconeogenic enzyme, was measured according to Harper (1965).The inorganic phosphate (Pi) released was determined as described by Fiske and Subbarow (1925).The concentrations of CP, DNA and RNA of LD were determined by the methods of Lowry et al. (1951), Labarca and Paigen (1980) and Munro and Fleck (1969), respectively.Plasma concentration of glucose and albumin were determined using commercial kits (Sentinel Diagnostics, Milan, Italy) and by bromocresol colorimetry (Roche Diagnostics GmBH, Mannhein, Germany, kit. no. 1970569).Plasma IGF-1 concentrations were determined by radioimmunoassay according to Louveau and Bonneau (1996) after formic acid and ethanol extraction.

Statistical analyses
All data were analysed using the SPSS software, vers.16.0 (SPSS, 2007).Because of the small number of the samples of sows, parity was not taken into account.
Sows reproductive traits, BFT, carcass traits, liver and LD composition and plasma concentrations of glucose, albumin and IGF-1 data were analysed using the general linear model (GLM) procedure with the one-way analysis of variance (ANOVA) using genotype as a fixed effect.The effects of gestation diet (access to acorns vs no access to acorns) on carcasses fatty acid composition were analysed according to a similar procedure using genotype and diet as fixed effects.Because birth weight has been reported to have an effect on body composition both between and withinlitter (De Passillé and Hartsock, 1979;Rehfeldt and Kuhn, 2006), data of body composition and blood plasma were adjusted by covariance using birth weight as covariate.
For plasma glucose concentrations, analysis of covariance was performed using genotype as a fixed effect and the time since the onset of farrowing as a covariate.For plasma albumin and IGF-1 concentrations, a GLM ANOVA was performed using two (light or heavy) and three (light, medium or heavy) birth weight classes for albumin and IGF-1, respectively, as a fixed effect.
Differences were considered significant at P < 0.05 whereas values between 0.05 and 0.10 were considered as trends.When adequate, means were separated by Tukey test.

Results
Backfat thickness at farrowing was not significantly different between genotypes (21.6 ± 1.3 mm vs 20.5 ± 1.4 mm for AL and LL sows, respectively; P > 0.05).Reproductive and productive traits of AL and LL sows are shown in Table 1.The AL sows had shorter gestation length (P < 0.001) than LL sows but no differences were detected between genotypes for farrowing duration.Litter size (total and born alive), litter weight of piglets born alive (P < 0.001) as well as individual birth weight (P < 0.02) were also lower in AL than in LL sows.Mortality rate (% born alive) between birth and 28d of age was higher in AL than in LL litters (25.1 vs 13.3%, P < 0.05).
The AL piglets used for the body composition study were lighter at birth and had lighter carcasses compared with LL piglets (1,181 ± 36 g vs 1,319 ± 36 g, P < 0.05 for birth weight and 953 ± 31 g vs 1,088 ± 31 g, P < 0.01 for carcasses).Data on carcass composition of piglets at birth are shown in Table 2.With the exception of ash, when adjusted for birth weight, carcasses of AL piglets had higher percentage of dry matter (P < 0.05) and protein (P < 0.01) than carcasses of LL piglets.The carcasses of AL piglets tended (P < 0.10) to have higher lipid content than carcasses of LL piglets.However, AL piglets had similar body fat content whether gestating sows had access to acorns.The LD of AL piglets contained more protein than the LD of LL piglets (95.4 ± 2.8 mg g -1 vs 79.7 ± 3.1 mg g -1 , P < 0.01).The difference tended to persist (P < 0.10) when data were adjusted for birth weight.However, DNA and RNA were unaffected by genotype even after adjustment for birth weight.In AL piglets, non-adjusted data for DNA and RNA were 1.77 ± 0.09 mg g -1 and 1.10 ± 0.04 mg g -1 , respectively.Corresponding values for LL piglets were 1.81 ± 0.10 mg g -1 and 1.05 ± 0.04 mg g -1 , respectively.There was a tendency for the RNA/protein ratio to be lower in AL piglets than in LL piglets (11.6 ± 0.6 µg g -1 vs 13.2 ± 0.7 µg g -1 , P < 0.10).Genotype had no effect on muscle glycogen concentrations averaging 8.8%.At the exception of fatty acids composition of the body lipids, there was no significant difference between piglets farrowed by AL sows having access or not to acorns during gestation.Data on fatty acid composition of the body lipids extracts are given in Table 3.When piglets born to sows exclusively fed the commercial diets (AL sows deprived of  acorns and LL sows) are compared, AL piglets had less C14:0 (P < 0.05) and C18:3 (P < 0.02).Within AL piglets, those born to sows having access to acorns during gestation had more C18:1 (P < 0.01) and less C16:0 (P < 0.02) than those farrowed by sows without access to acorns.Data on liver weights, glycogen contents and G6-Pase activity are presented in Table 4. Genotype has no significant effect on the absolute weight of the liver.However, relative to birth weight, liver was 12.1% (P < 0.05) heavier in AL piglets.No significant difference between genotypes was observed on hepatic glycogen contents or G6-Pase activity.
In both genotypes, blood glucose concentration was not dependent on birth weight, but was positively correlated with the time elapsed since the onset of farrowing (r = 0.276; P < 0.001).Values adjusted for the time since the onset of farrowing were 637 ± 22 and 541 ± 19 mg mL -1 (P < 0.01) in AL and LL piglets, respectively.
Plasma albumin concentrations adjusted to birth weight averaged 6.6 ± 0.3 mg mL -1 in AL piglets that was higher (P < 0.05) than the 5.5 ± 0.3 mg mL -1 found in LL piglets.Values for plasma albumin concentrations of light (1,026 ± 35 g) and heavy (1,288 ± 31 g) AL piglets were 5.2 ± 0.5 and 7.1 ± 0.4 mg mL -1 (P < 0.01), respectively.In LL piglets no significant differences were observed between birth weight categories.In AL but not in LL piglets, there was a positive within-litter correlation between plasma albumin concentrations and birth weight (r = 0.34; P < 0.01).
of feeding, i.e., 0.45 kg d -1 from d 85 to farrowing (Ojamaa et al., 1980), or a severe protein deficiency (Pond et al., 1969), most of the studies (Yen et al., 1982;Hausman et al., 1991;Ruwe et al., 1991) reported no significant influence of gestational feeding (frequency, quantity, composition) both on birth weight or body composition of newborn piglets.The only exception is the fatty acids composition that can be influenced by the gestational feeding composition in fatty acids (Gerfault et al., 1999;Rooke et al., 2001) as has been observed in the current trial.Therefore, the observed differences between newborn of the two genotypes studied, should be largely attributed to differences in production systems (genotype + environment) and not to the different feeding regimens.Also, despite differences in feeding quantities and physical activity, body condition at farrowing of AL sows, estimated from the depth of subcutaneous fat just after farrowing was similar to that of LL sows.
On the whole, reproductive performance results of AL sows in this experiment are largely similar to those reported by Charneca (2001) and by Marques (2001).Briefly, AL sows are less prolific than LL sows, have a lower duration of gestation (Nunes, 1993) while piglets are lighter at birth and present a higher mortality rate until weaning.
Whereas carcass chemical composition of the LL piglets agrees with those previously reported for this genotype (Herpin et al., 1993;Canario et al., 2007b), results of this study indicate that AL piglets differ markedly at birth from LL piglets on body composition, plasma glucose, albumin and IGF-1 concentrations.After adjustment for birth weight, the AL newborns possess more dry matter and protein contents than LL piglets while body fat only tends to be higher in AL piglets.It is relevant to notice that despite the capacity for protein synthesis estimated from the RNA/protein ratio (Attaix et al., 1988) tended to be lower in AL piglets, the percentage of body protein was higher than in LL piglets.This and the modest difference in the percentage of body fat between AL and LL piglets are the reverse of what has been reported for these pigs at seven weeks of age (Freire et al., 1998) and older (Nunes, 1993;Nieto et al., 2002).Perhaps, as in the Meishan pig (Bonneau et al., 1990), protein deposition in older AL pigs is limited by the number of muscle fibers which is fixed at birth.The Meishan pig has less muscle fibers at birth.This remains to be determined in AL piglet.However that may be, present results are largely similar to those reported when foetuses from obese and lean strains of pigs (Hoffman et al., 1983;Stone et al., 1985) and, newborns from Meishan breed and those from a line highly selected for muscle growth are compared (Herpin et al., 1993).Genetic selection on lean growth and litter size from 1977 to 1998 also resulted in similar differences in the body composition of the newborns (Canario et al., 2007b).
Although total body fat content at birth was similar in AL piglets, those born from sows which consume acorns had more C18:1 in their body fat than those born to AL sows deprived of acorns or LL piglets.Acorns contain high level of C18:1 (higher than 60% of fatty acids, Rey et al., 1997).This would suggest that some dietary fatty acids could cross the porcine placenta, in agreement with Gerfault et al. (1999) and Rooke et al. (2001).Further, colostrum from sows fed acorns during gestation is found to contain more fat with a higher proportion of C18:1 (Charneca, 2001).These and the fact that oleic acid is the most readily fatty acid oxidized by the newborn pig (Schmidt and Herpin, 1998) suggest that feeding acorns to gestating sows would be of interest for the energy metabolism of the newborn pig.
In this study, AL piglets had relative heavier livers but there was no significant effect of genotype on liver glycogen stores.Indeed, hepatic gluconeogenic capacity is not affected by genotype as illustrated by the activity of the G6-Pase.Further, muscle glycogen concentration is not affected by genotype.Therefore, despite a 4 days difference in gestation length, glycogen reserves are similar in both genotypes.These results agree with those reported when comparing piglets born from obese Meishan sows to those born from a line highly selected for muscle growth (Herpin et al., 1993) while genetic selection for lean growth and litter size from 1977 to 1998 also resulted in a decrease in the relative weight of the liver (Canario et al., 2007b).
There was an effect of genotype on plasma glucose, albumin and IGF-1 concentrations, all being higher in AL piglets.These results agree with what has been observed when comparing piglets born to obese Meishan sows to those born to a line highly selected for muscle growth (Herpin et al., 1992(Herpin et al., , 1993)).Genetic selection for lean growth and litter size from 1977 to 1998 also resulted in similar changes in plasma parameters (Canario et al., 2007b).Plasma glucose concentrations are variable at birth, increasing during the course of parturition (Herpin et al., 1996).However, in this study data were adjusted to birth time.There-fore, the higher plasma glucose concentrations found in AL piglets might be a trait of this breed.In some studies, the within-litter plasma albumin (Stone and Christenson, 1982;Wise et al., 1991) and IGF-1 concentrations (Herpin et al., 1992) are positively related to birth weight.In the present study only the withinlitter plasma concentration of albumin was positively correlated to birth weight in AL piglets, suggesting that plasma albumin and IGF-1 concentrations are not invariably correlated with birth weight.
Overall, both vital deposition of energy reserves and chemical body composition are not impaired in Alentejano newborns despite a shorter gestation length.Further, increased body protein at birth, relative liver weight (Herpin et al., 1993;Canario et al., 2007b) and plasma albumin (Stone and Christenson, 1982;Wise et al., 1991) are considered as good indexes of development and maturity.Similarly, level of plasma IGF-1 is considered to be indicative of the maturity of the IGF axis (Greenwood et al., 2002).On these bases, piglets born from Alentejano sows are expected to be more mature at birth than those born from conventional sows.Further, similar to the Meishan female (Legault and Caritez, 1983), the onset of puberty occurs earlier in the IB female (González-Añover et al., 2009) than in modern commercial crosses (LW × LR) when fed similarly.This and the greater maturity at birth would indicate that the IB swine is more precocious than conventional swine.Genetic selection for lean growth and litter size from 1977 to 1998 also resulted in a shorter, but to a lesser extend, duration of gestation (Canario et al., 2007a).However, this shorter gestation length was associated with a less maturation of piglets (Canario et al., 2007b).Therefore, a shorter gestation length may be specific for the Alentejano breed.
In conclusion, the shorter gestation lenght of AL sows has no negative effect on the energy stores of the newborn.However, the body protein content at birth is markedly higher in AL than in LL piglets suggesting that studies are required to determine the muscle histochemical and biochemical charateristics at birth.This would help to understand the low potential of the growing animal to deposit protein.Further, the reduction of piglet mortality is an important goal during the neonatal period.In this respect, future research should focus on the early nutrition (colostrum consumption) and the acquisition of passive immunity of piglets as well on the mothering ability of the sows.

Table 1 .
Alentejano (AL) and Large-White × Landrace (LL) sows reproductive and productive traits Each value represents the least squares mean ± SEM for each genotype. 1Live born piglets.

Table 2 .
Carcass composition at birth of Alentejano (AL) and Large White × Landrace piglets (LL) 1 1Measurements were made on 12 piglets per genotype at the exception of ash (7 piglets per genotype).

Table 3 .
Fatty acid composition of lipid fraction in carcasses of AL (Alentejano breed) and LL (Large White × Landrace crossbred) piglets at birth Each value represents the least squares mean ± SEM for each genotype. 1% of the identified fatty acids.