In both humans and animal species, Intrauterine Growth Restriction (IUGR) and subsequent Low Birth Weight (LBW) is a common feature in pregnancies challenged by either maternal malnutrition/hypoxia or placental insufficiency [1
]. Offspring with LBW is affected by increased perinatal mortality and morbidity in the short-term and by decreased growth patterns and health status and performance in the long-term [2
Possible strategies for preventing or alleviating the occurrence of IUGR are based on maternal supplementation with antioxidant agents [3
], since the IUGR condition is characterized by a weakened antioxidant defense system of the fetuses [4
]. Polyphenols are potent antioxidant agents and the most abundant dietary antioxidants [6
]. Our group has proven the usefulness of maternal supplementation with hydroxytyrosol (a polyphenol present in olive leaves and fruits [7
] with even higher antioxidant capacity than vitamin E [9
]) for improving fetal oxidative status and decreasing the appearance of LBW neonates in a swine model of IUGR [10
Polyphenols are well-considered and freely consumed by the general population because they are perceived as “natural” products for the prevention and treatment of different diseases. In the same way, there is a strong trend for its use during pregnancy and, as an example, 78% of the pregnant women in United States of America take supplements during pregnancy [12
]. However, there is a scarcity of data from studies evaluating the practical benefits and potential hazards of polyphenols supplementation during pregnancy [13
]. Hence, long-term preclinical studies are necessary to determine the real effects of maternal polyphenol supplementation on the postnatal development and the health/disease status of the offspring. We have previously studied the effects from hydroxytyrosol on prenatal and early postnatal (lactation) development. The current study aimed to increase the available information by determining the effects of the hydroxytyrosol prenatal supplementation on the growth patterns, metabolism and body composition in our swine IUGR model during later stages, during the juvenile development.
2. Materials and Methods
2.1. Animals and Experimental Procedure
The study was carried out at the INIA animal facilities according to the EU Directive 2010/63/UE for the protection of animals used for scientific purposes. The experimental procedures were specifically assessed and approved by the INIA Committee of Ethics in Animal Research (report CEEA 2013/036).
The experiment involved 93 Iberian piglets born from 18 pregnant sows which, at Day 35 of pregnancy, were pair-matched according to body weight in two equal groups (control and treated with hydroxytyrosol; n
= 9 each one). These sows were fed with a diet (further data in Table S1
) fulfilling 50% of daily maintenance requirements from Day 35 of gestation until delivery [14
] to impose a nutritional challenge inducing IUGR in the offspring [15
]. Nine of them (group HT) acted as the treated group by receiving 1.5 mg of hydroxytyrosol per kg of feed per day while the other nine sows remained as the untreated control group (group C).
After birth, piglets remained with sows until weaning at 25 days-old. At weaning, 41 piglets (10 females and 11 males from the group C and 10 females and 10 males from the group HT), representative of average weight and size of the breed and similar between both groups, were chosen from different litters for avoiding the effects of sow and birth-weight. Currently, it is well-known that weaning-weight has an ineludible effect on postnatal development [17
] and, for avoiding masking effects of this variable on the results of the study, we selected piglets with similar weaning-weights. Since the maternal hydroxytyrosol supplementation might improve the development during lactation [10
], this design allow properly assessing its effect on postnatal development only after weaning. After weaning, the piglets were fed with two standards diets adapted to age (26–60 and 60–180 days-old; further data in Table S1
). The amount of feed offered was re-calculated with age for fulfilling daily maintenance requirements. One female from each group died during the experimental period, so 39 pigs were finally used for the study. In these pigs, we studied the effects of the maternal hydroxytyrosol supplementation on postnatal changes in body weight and size, adiposity, plasma indexes of glucose and lipid metabolism, histological characteristics of muscle and fat, and tissue fatty acid (FA) composition after weaning. From the results obtained analyzing fatty acid composition, the biobanked organs of 52 piglets which were born from the same litters and sampled at weaning (6 females and 11 males from the group C and 10 females and 25 males from the group HT) were used for determining the FA composition.
2.2. Evaluation of Growth Patterns, Corpulence and Adiposity during Juvenile Development
In all pigs, body weight and size (occipito-nasal length, biparietal diameter, trunk length, maximum thoracic diameter, and abdominal and thoracic circumferences) were recorded from weaning to 180 days of age (average of 25, 45, 60, 75, 90 120, 150 and 180 days-old). The body weight values were used to calculate average daily weight gain (ADWG) in the intermediate periods of age and for lifetime. Concomitantly, subcutaneous back fat depth (divided into outer and inner layers and total back fat depth) and longissimus dorsi diameter were determined at the P2 point (a point at the right side of the animal located at 4 cm from the midline and transversal to the head of the last rib) using a SonoSite S-Series ultrasound machine with a lineal array probe (5–8 MHz; SonoSite Inc., USA).
2.3. Evaluation of Blood Indexes of Carbohydrate and Lipid Metabolism during Juvenile Development
At 120, 150 and 180 days-old, after fasting for approximately 16 h, blood samples were drawn from the orbital sinus using EDTA vacuum tubes (Vacutainer Systems Europe, France). Samples were immediately centrifuged at 1500 g for 15 min, and plasma was separated and stored at −20 ℃ until analysis. Parameters of glucose (glucose and fructosamine) and lipid profile (total cholesterol, high-density lipoprotein cholesterol [HDL-c], low-density lipoprotein cholesterol [LDL-c], and triglycerides) were measured by a clinical chemistry analyzer (Saturno 300 plus, Crony Instruments SRL, Rome, Italy), according to the manufacturer’s instructions.
2.4. Evaluation of Organ Weights and Histological Characteristics of Muscle and Fat at 180 Days-Old
The pigs were sequentially euthanized by stunning and exsanguination at 180 days-old, in compliance with RD53/2013 standard procedures. Immediately, the weights of head, carcass and total and individual viscerae (adrenal glands, brain, heart, intestine, kidneys, liver, lungs, pancreas and spleen) were determined. The weight-ratios of carcass and individual viscerae to body weight were also calculated and expressed by percentage.
Two samples of muscle (from longissimus dorsi
at the level of last rib and gluteus medius
) and subcutaneous back fat were obtained. Immediately, one of them was stored at −20 °C for assessment of total fat percentage and fatty acids composition while the other was fixed in 10% neutral-buffered-formalin and processed for the evaluation of number of muscle fibers and adipocytes after hematoxylin-eosin staining (Figure 1
). Total fat percentage and FA composition were also determined in samples from the brain and the right lateral lobe of the liver. One biobanked sample of longissimus dorsi
, gluteus medius
, liver and brain from piglets slaughtered at 25 days-old were also used to analyze fatty acid composition.
The third sample of longissimus dorsi
was used to determine drip-loss capacity on the same day of sampling [18
] and meat analysis. For instrumental meat color measures, samples of 2 cm2
of area were kept at 4 °C. Color was evaluated on days 0, 3 and 6 postmortem by a spectophotometer (CM-600d, Konika Minolta Sensing Inc., Camera, Japan), previously calibrated with the manufacturer’s instructions. Aperture was set to 10° and light source to D65. The average of three random readings was used to measure lightness (L*), redness (a*), and yellowness (b*) by CIE L*a*b* values and the amount of myoglobin chemical forms was calculated via K/S ratios [19
2.5. Evaluation of Fatty Acid Composition in Diet and Pigs at 25 and 180 Days-Old
Fatty acids in the feed were identified and quantified after extraction and methylation [20
], by gas chromatography (HP6890, Hewlett Packard, Avondale, PA, USA) with a capillary column (30 m × 0.32 mm i.d. and 0.25 µm polyethylene glycol-film thickness, HP-Innowax) and a flame ionization detector [21
]. The results obtained are detailed in Table S1
The lipids from intramuscular fat, brain, and liver were extracted (expressed as a dry matter percentage; [22
]) and fractionated into the neutral lipid (triglycerides) and polar lipid fractions (phospholipids) [23
]. The fatty acids of the subcutaneous back fat were separately analyzed in outer and inner layers. Finally, lipids were methylated and identified [24
]. The individual fatty acid percentages were used to calculate proportions of saturated, monounsaturated and polyunsaturated fatty acids (SFA, MUFA and PUFA, respectively), the unsaturation index (UI), and the sum of total ∑n-3 fatty acids and ∑n-6 fatty acids and their ratio (∑n-6/∑n-3 fatty acids) [25
]. Moreover the activity of the stearoyl-CoA desaturase enzyme 1 was estimated as desaturation indexes (SCD1; the ratio of the enzyme product, MUFA mainly oleic acid [C18:1n-9], to the enzyme substrate, SFA mainly stearic acid [C18:0]), as previously described [26
2.6. Statistical Analysis
Data were analyzed by the SAS version 9.4 (Statistical Analysis System Institute Inc., USA). First, births were categorized in small (< 8 piglets) and large litters (≥8 piglets), according to Iberian breed data, to analyze the effect of litter size. After testing the normality of data using a Shapiro-Wilk test, dependent variables were assessed using three-way ANOVA in a general linear model including maternal gestational treatment (C vs. HT), sex (female vs. male) and litter size (< 8 vs. ≥ 8 piglets/litter), and all maternal treatment interactions. Dependent variables with changes over time were also assessed by repeated measures ANOVA with the Greenhouse-Geisser correction. Sow was used as a random effect in the weaning analysis to account for the common maternal environment. For performance parameters, the respective age was used as a covariate. Mean values were expressed by estimated marginal means with the different effects included for each variable. The pig was the experimental unit, and the statistical significance was accepted from p < 0.05. All the results were expressed as mean ± S.E.M.
The results from the present study indicate that a maternal supplementation with hydroxytyrosol during pregnancy has long-lasting effects during the postnatal life of the offspring. In brief, from equal body weight at weaning, pigs from sows treated with hydroxytyrosol had greater growth, body weight, corpulence (wider thoracic and abdominal circumferences), muscle development and adiposity (higher thickness of total and inner layer of subcutaneous back fat) than their control counterparts. Conversely, the main effects on metabolism and body composition were not found, except in changes in the fatty acid composition of pigs from large litters, those more affected by IUGR processes. Hence, the join results of previous research and the current trial suggest that maternal supplementation with hydroxytyrosol improves pre- and early post-natal development of offspring [10
] but also juvenile development.
We need to highlight that the data obtained in the present studied were the result of comparing offspring from sows exposed to undernutrition during the pregnancy, either with or without hydroxytyrosol supplementation, without establishing a control group fed ad libitum
which would allowed to compare the effects of hydroxytyrosol supplementation during restriction with traits in animals without restriction. These obviously is a limitation to the interpretation of the results but the restriction model was established previously and, in the current trial, we preferred to avoid an excessive use of animals in agreement with 3Rs [10
It is also highly important to emphasize that the piglets used in the present study had the same body weight in both the control and treated groups at the beginning of the study (at weaning). Afterwards, individuals from pregnancies supplemented with hydroxytyrosol had a higher ADWG and therefore reached a higher body weight than controls as soon as during the early post-weaning phase (25-75 days-old). The development during the post-weaning phases is strongly related to the growth during lactation and afterwards the body weight at weaning [17
]; characteristics which have been found to be improved by a maternal hydroxytyrosol supplementation [10
]. However, due to the selection of individuals with average and similar body weights, the findings of the present study were independent of the positive effects of prenatal hydroxytyrosol supplementation on birth weight and body development during lactation previously described [10
]. Therefore, they could be related to positive effects on physiology and metabolism. There is evidence addressing that the prenatal use of polyphenols may increase epigenetic changes, through DNA methylation, histone modifications and/or variations in the miRNA expression [27
]. In this sense, a previous study of our group showed that maternal hydroxytyrosol supplementation improved the fetal antioxidant status and prevented possible DNA hypomethylation associated with oxidative stress during the stages concurrent with the maternal treatment [11
Afterwards, during the last period of the juvenile phase (from 120 to 180 days-old), the maternal hydroxytyrosol supplementation was also found to be related to significant increases in body weight but also corpulence. Increases in body weight and corpulence may be considered a positive effect of the treatment, mainly because of their association with greater muscle development. However, we should also have in mind that they were also related to higher adiposity. First, for pigs and minipigs, thoracic circumference has been found predictive for the amount of carcass fat while abdominal circumference would be predictive for the amount of visceral and subcutaneous fat [29
]. Second, because of a higher thickness of subcutaneous back fat.
The increase in the subcutaneous back fat depth of the treated group was mainly caused by an increase in its inner layer. The outer layer is more related to protection from cold climates while the inner layer is metabolically more active, mainly due to a high lipoprotein lipase activity [32
]. Previous studies have linked the activity of lipoprotein lipase to obesity and insulin resistance [33
]; however, there were not found critical changes in the amount of visceral and intramuscular fat or the parameters of glucose and lipid metabolism in the pigs from hydroxytyrosol-treated pregnancies. There were only found higher fructosamine concentrations at 150 days-old, which reflects higher glucose values during the previous weeks. The assessment of fatty acids composition showed that treated pigs had a lower desaturation index in the outer layer of the subcutaneous fat at 180 days-old, which is indicative of a good metabolic status [34
]. Hence, although we did not found critical changes in metabolism, the evidence of a higher adiposity in the offspring from sows treated with hydroxytyrosol need further consideration and support the necessity of long-term safety data for the use of the compound during pregnancy; having also in mind the possible effects of prenatal polyphenol use on obesogenic gene expression [36
The results of the current study have a dual value, as a translational animal model for biomedical studies but also for swine production. From the point of view of animal production, the nutritional supplementation of pregnant sows with hydroxytyrosol is highly interesting, since it improves ADWG and growth patterns, giving way to animals with higher body weight and greater muscle development without increasing the weight of internal organs. The assessment of meat quality, in terms of fatty acids composition, showed a lower ∑n-6 content in pigs of large litters from hydroxytyrosol supplemented sows, which may also be useful if considering the human diet recommendations advising to reduce ∑n-6 intake [37
]. Data also supports a better meat quality at earlier life stages, when higher desaturation indexes were also found in the muscles of weaning piglets [40
]. On the other hand, we have to note that, despite a higher diameter of the longissimus dorsi
in treated pigs, there were no differences in the number of muscle fibers. Hence, the increase in the size of muscle is related to processes of fiber hypertrophy which may mean changes in meat quality traits [43
]. Although the present results did not indicate major changes in meat morphological characteristics, such findings deserve further research addressing possible changes in meat organoleptic traits.
Finally, it is important to highlight that, contrary to previous findings in this model [10
], there were not found any significant effect from offspring sex in growth patterns; such extent may be related with the fact that males were castrated at birth for their management. It is also important to note that pigs from small litters had overall better growth patterns and meat quality characteristics. There were also less pronounced effects from maternal hydroxytyrosol treatment in pigs from small litters than in pigs from large litters. These results are in agreement with previous data addressing a higher incidence of IUGR processes and therefore a higher number of LBW piglets in large litters in both lean and fatty pigs, which compromises postnatal development [10
]. However, we need to have in mind that all the piglets in the current experiment had the same weaning body weight, just for avoiding effects of LBW and deficiencies in early postnatal development. Hence, the present trial is suggesting that other effects from litter size may set the basis for further research.
In conclusion, the novel findings of the positive effect of maternal hydroxytyrosol supplementation on postnatal development and meat quality described in the present article show hydroxytyrosol as a useful nutritional tool to improve offspring traits. However, further studies are necessary to elucidate possible effects on adiposity trends and metabolic status, and their real translational value for human beings, and meat characteristics when implemented for improving pork production.