Sows undergo placenta formation, rapid fetal development, parturition and lactation during the reproductive cycle. During late gestation, the rapid growth and development of the fetus results in the mother maintaining a high catabolic state [1
]. In addition to their diet, sows break down their own fat for their fetuses [2
]. After delivery, sows maintain a catabolic state due to the need for synthetic milk [3
]. Under normal conditions, cell oxidative metabolism will generate a small amount of reactive oxygen species (ROS). However, during late gestation and lactation, the greatly increased metabolic intensity of sows can induce oxidative stress [4
]. This process is characterized by a large accumulation of ROS in the body, including large amounts of superoxide and hydrogen peroxide produced by the placenta and mammary gland [5
]. Oxidative stress occurs when the amount of ROS produced in the body exceeds the neutralizing capacity of antioxidant enzymes such as glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) [6
]. Excessive ROS can cause oxidative damage to proteins, lipids and DNA, ultimately disrupting cellular function. It has been shown that sows have elevated levels of oxidative stress markers ROS, thiobarbituric acid reactants and 8-hydroxy-2 deoxyguanosine in serum during late gestation and early lactation [2
]. Oxidative stress in sows not only reduces reproductive performance but also leads to reduced feed intake, prolonged negative energy balance, deterioration of body status and reduced milk production during lactation [8
]. Therefore, it is important to develop effective nutritional interventions for oxidative stress in sows.
Amino acids (AA), as the basic material of protein for animal nutrition, are actively involved in a series of physiological processes such as protein metabolism, nitrogen balance and enzyme synthesis [9
]. Many studies have shown that amino acids have an antioxidant function and can relieve the oxidative stress of the body. Glutamine (Gln) is a rate-limiting precursor for the synthesis of glutathione, an important antioxidant in the body [11
]. Maternal Gln supplementation could improve the intestinal development of piglets by inhibiting the expression of miR-29a and promoting the expression of the tight junction protein [12
]. Supplementation with Gln could increase the content of SOD and decrease the content of malonaldehyde (MDA) in the serum of weaned piglets [14
]. Leucine (Leu) is an essential and directly linked amino acid (BCAA) whose physiological functions include regulating protein metabolism, providing oxidative energy and enhancing antioxidant enzyme activity [15
]. Maternal Leu supplementation could promote skeletal muscle protein synthesis and alleviate intrauterine growth restriction in fetuses through mTOR/S6 K1 pathways [17
]. A study has shown that Leu supplementation can increase the expression of antioxidant genes in the liver of finishing pigs under heat stress, which may be related to the Keap1-Nrf2 signaling pathway [19
]. Gamma-aminobutyric (γ-GABA), as a natural non-protein amino acid, can significantly increase the content of GSH-PX in serum and reduce the levels of MDA and adrenocortical hormone, thereby alleviating transport stress in growing–finishing pigs [20
]. Therefore, amino acids have potential application value in reducing oxidative stress and improving productive performance in sows.
Diets supplemented with Gln, Leu or γ-GABA, individually, were reported to be effective in improving the performance of pigs [12
]. However, it remains to be explored if different combinations of these three amino acids are effective or not for reducing oxidative stress in sows and enhancing reproductive performances. The present study aimed to investigate the effects of maternal dietary supplemented with Gln, Leu and γ-GABA combinations from late gestation to the end of lactation on performance and the antioxidant and immunological status of sows and their piglets.
2. Materials and Methods
L-glutamine and L-leucine were purchased from Henan Mengzhide Trading Co., Ltd., Zhengzhou, China. γ-aminobutyric acid was purchased from Suzhou Lejuan Biotechnology Co., Ltd., Suzhou, China. L-alanine (Ala) was purchased from Hebei Huayang Biotechnology Co., Ltd., Hengshui, China.
2.2. Animals and Treatments
Fifty large white × landrace sows (average BW, 182.94 ± 3.04 kg; parity, 1.11 ± 0.38) on day 85 of gestation were divided into five groups, with ten sows in each group, according to the principle of similar BW and parity. The basal diet was formulated on the basis of the nutritional needs of pregnant and lactating sows according to the NRC 2012 (Table 1
). Based on the basal diet, the control group was supplemented with 1.05% Ala, and the other four groups were supplemented with AAMs1 (basal diet + 0.55% Gln + 0.44% Leu + 0.01% Ala), AAMs2 (basal diet +0.55% Gln + 0.01% GABA + 0.29% Ala), AAMs3 (basal diet + 0.44% Leu + 0.01% GABA + 0.67% Ala) and AAMs4 (basal diet + 0.55% Gln + 0.44% Leu + 0.01% GABA), respectively. Treatments were started from day 85 of gestation to day 21 of lactation. Sows were housed individually in pregnant house from day 85 to 109 of gestation and transferred to the farrowing house individually on day 110 of gestation until weaning on day 21 of lactation. Each sow was kept in separate pens. The temperature of the farrowing room was strictly controlled at 20 °C, and the room light cycle was set at 06:00 to 16:00. Sows during late gestation were restricted to a 3.5 kg daily diet. The sows had ad libitum access to feed and water during the entire lactation period. The total daily ration and feed intake for each sow were recorded separately. Piglets were provided free access to milk and treated according to routine management. Creep feed was supplied to piglets on day 8 postpartum. All sows were delivered within two weeks. The neonatal piglets were weighed within 12 h after birth. Litters were standardized to achieve an approximately similar number of suckling piglets per litter by cross-fostering within each group.
2.3. Litter Performance and the Feed Intake of Sows
Litter size at birth, birth weight and litter birth weight were used to evaluate the litter performance of sows. Feed intake during lactation was recorded.
2.4. Growth Performances of Neonatal Piglets
Average litter weight and average body weight on days 7, 14 and 21 after birth were recorded and calculated for the ADG and average daily litter gain (ADLG).
2.5. Sample Collection
Blood samples were collected from sows and piglets on day 110 of gestation and days 7, 14 and 21 of lactation, respectively. Six piglets were sampled from different litters in each treatment group according to the average body weight. The serum was separated by centrifugation at 3000 × rpm for 15 min and stored in 1.5 mL aliquots at −80 °C. Within 5 min of parturition, a total of 25 mL of colostrum was extruded from the front, middle and rear teats of sows and stored at −20 °C.
2.6. Determination of Amino Acid
The concentrations of free amino acids in serum and colostrum were measured by ultra-high-performance liquid chromatography. Samples were centrifuged at 5000 × rpm for 5 min at 4 °C. 200 μL supernatant was transferred to 1.5 mL centrifuge tube. Thereafter, 8 μL of phenylpropanoid solution and 800 μL of methanol were added in sequence and kept at 4 °C for 2 h. The solution was centrifuged at 14,000 rpm for 10 min at 4 °C, and then 200 μL of the supernatant was collected and dried at 55 °C for 3 h. A total of 100 μL boric acid was added to dissolve the dried sample. A total of 10 μL of dissolved sample was mixed with 50 μL boric acid buffer and 20 μL derivatizing reagent. After one minute, the tube was sealed and heated at 55 °C for 10 min, and the content of free amino acids was determined by ultra-high-performance liquid chromatography (Agilent1200, Agilent, Santa Clara, CA, USA).
2.7. Assessment of Colostrum Composition
Milk fat, lactoprotein, lactose and total solids were assessed by a Milk Component Somatic Cell Count Tester (YQ1-55, Bentley Instruments, Chaska, MN, USA). Immunoglobulin A (IgA), Immunoglobulin G (IgG) and Immunoglobulin M (IgM) of colostrum were tested by ELISA assay kits (Prodia Diagnostics Company, Bötzingen, Germany). Total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and GSH-PX and MDA levels in serum were analyzed by assay kits (Nanjing Jiancheng Biotechnology Company, Nanjing, China). T-AOC was assessed by the ABTS method. T-SOD was assessed by the hydroxylamine method. GSH-PX was assessed by an assay kit with the colorimetric method. The MDA concentration was assessed by an assay kit with the thiobarbituric acid method. Before analyzing antioxidant indicators, we removed the cellular component of the colostrum by cryogenic centrifugation at 3000 × rpm for 15 min.
2.8. Determination of Serum Biochemical Parameters
Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined by using an Automatic Biochemical Detector (Hitachi 7600, Hitachi, Tokyo, Japan). IgG and IgM were tested by using assay kit from Prodia Diagnostics Company, German. Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) were measured by using ELISA test kits (Beijing Konka Hongyuan Biotechnology Co., Ltd., Beijing, China). D-lactate (D-LA) in serum were tested by using assay kits from Nanjing Jiancheng Biotechnology Company, China. T-AOC, T-SOD, GSH-PX and MDA levels in serum were analyzed by assay kits (Nanjing Jiancheng Biotechnology Company, Nanjing, China).
2.9. Statistical Analysis
Data were analyzed statistically using SPSS 21.0 (Chicago, IL, USA). Individual sows and total piglets in a litter were respectively regarded as an experimental unit to analyze the litter performances of sows and the growth performance of piglets. Six piglets from different pens per group close to the group average body weight were considered as the unit of analyses for the difference in serum indicators. One-way ANOVA and Duncan test were used to analyze the significance of differences between groups for data satisfying a normal distribution, and a nonparametric test was used to analyze the significance of differences between groups for data not satisfying a normal distribution. Results are expressed as means ± SEM. Probability values less than 0.05 indicate a significant difference, and probability values between 0.05 and 0.1 indicate a trend.
The metabolic intensity of sows increases greatly during late gestation, which causes the imbalance of the redox system in sows and then affects their reproductive performance and the growth of offspring. The present study demonstrated that maternal AAMs supplementation improved the reproductive performance of sows and the growth performance of piglets. The results further showed that these beneficial effects are mediated by the improved colostrum composition and enhanced antioxidant capacity.
Sows during late gestation have intensive metabolism and mobilization, producing a large number of oxygen free radicals that destroys the balance of the body’s oxidation reduction system [4
], which can be subsequently associated with the intrauterine growth restriction of the fetus [23
]. SOD and GSH-PX play important roles in eliminating excess oxygen free radicals and reducing oxidative damage in animals [23
]. Leu could activate the Nrf2-related signaling pathway, thus promoting the expression of antioxidant enzymes SOD2, catalase and heme oxygenase-1 [24
]. Dietary Gln supplementation could improve the resistance of broiler muscle to oxidative damage through the expression of the Nrf2-Keap1 signaling pathway [25
]. Maternal GABA supplementation could upregulate the GABA receptor, thus enhancing the SOD activity of sows and alleviate placental oxidative stress [26
]. In the present study, higher levels of serum T-AOC, T-SOD and GSH-PX in AAMs groups on day 110 of gestation were observed, indicating the antioxidant capacity of sows was improved. Studies have shown that the enhancement of maternal antioxidant capacity is beneficial for the growth and development of offspring [8
]. At the same time, we also observed that the serum amino acid including Leu, Glu and Lys concentrations of sows supplemented with AAMs were increased. Amino acids are crucial to the development of the fetus, and most amino acids are supplied from the maternal circulation to the fetuses by activating transport across the placenta [27
]. Higher serum free AA concentrations of sows in AAMs groups on day 110 of gestation suggest increasing AA supply for fetuses. Leu has been shown to alter placental metabolism and promote maternal–fetal nutrient transport via PI3K/AKT/mTOR signaling pathway [28
]. Glu provides a precursor for the synthesis of GSH-PX, which is involved in antioxidant reactions [29
]. Therefore, we inferred that the improvement of antioxidant capacity and the increase of AA supply in sows during late gestation not only improved the maternal physiological status but also enhanced the litter performance to a certain extent, which was reflected in the increasing trend of healthy litter numbers in the AAMs groups.
Although the effects of AAMs supplementation on the litter performance of sows was not significant, the improvement in the growth performance of piglets in the AAMs group during lactation was noteworthy. In this study, all AAMs supplementation increased the growth performance of piglets. Among them, the combination of Gln, leu and γ-GABA had the best growth-promoting effect. Interestingly, it has been demonstrated that supplementing Gln in a rabbit diet had no positive effect on the litter weight at weaning [30
]. There was no significant change in the average daily gain of weaned male pigs supplemented with γ-GABA for 35 days [31
]. There was no effect on the growth performance of broilers fed with Leu [32
]. It might suggest that AAMs supplementation can easier benefit piglet growth performance compared to supplementing with single AA.
Next, we explored the potential pathways of AAMs supplementation to improve growth performances in piglets. The growth of animals is largely affected by nutritional status and hormonal activity [21
]. Breast milk is the main nutrient source for piglets during the neonatal period. In our study, maternal AAMs supplementation enhanced the colostrum lactoprotein, lactose and total solid contents, meaning more available nutrients for neonatal piglets. Milk protein synthesis by the mammary gland requires the absorption of large amounts of amino acids from the blood. The requirements for Leu, Arg and Lys reached more than 30 g/d [33
]. Our results showed that all AAMs supplementation increased serum Lys levels in sows and Leu + γ-GABA supplementation increased serum Arg level. Lys is the first limiting amino acid of lactating sows [35
]. Therefore, the increase in the Lys level may be an important reason for the increase of milk protein content. In addition, Arg can improve lactation performance by enhancing mammary tissue growth and nutrient uptake through the production of NO, a major vasodilator and angiogenic factor [36
]. Growth and development are also regulated by GH and IGF-1. The serum levels of GH and IGF-1 were found to be positively correlated with the growth of the piglets [21
]. In our study, higher serum GH and IGF-1 levels in AAMs groups indicated that these piglets had higher anabolic activity during lactation and can promote their growth and development.
Birth oxidative stress is an oxidative response to a sudden transition process from maternal mediated respiration in the uterus to autonomous pulmonary respiration outside the uterus. It is confirmed that piglets suffer from heavy oxidative stress after birth, which causes oxidative damage to lipids, proteins and DNA [37
]. AAMs supplementation can increase the level of T-AOC in the colostrum and serum of piglets, which is helpful for neonatal piglets to resist the adverse effects of oxidative damage. In addition, after the piglets are born, microbes begin to colonize the intestine. The incomplete development of intestinal function in the newborn stage is prone to the invasion of pathogenic microorganisms, which can cause intestinal barrier and liver damage [38
]. Therefore, neonatal piglets must acquire maternal immunoglobulins from the colostrum for passive immune protection [40
]. Gln is an important energy and biosynthetic nutrient for the proliferation, survival and function of B cells, which are important immunoglobulin producers [41
]. A study has reported that GABA supplementation can also increase serum immunoglobulin content in hens. The probable reason is the inhibition of GABA on somatostatin and adrenal corticosteroid hormone secretion [43
], which impairs immunoglobulin. Therefore, dietary AAMs supplementation could enhance the IgA and IgG levels in colostrum, thereby helping piglets obtain passive immunity. IgA plays an important role in regulating intestinal microbiota and preventing early intestinal inflammation, which could distribute in mucosa to prevent the invasion of pathogenic microorganisms after entering the intestine [44
]. IgG largely recognizes virulence factors encoded within the locus of the enterocyte effacement pathogenicity island, including the adhesin Intimin and T3SS filament EspA, which are major antigens conferring protection [45
]. Thus, IgG in breast milk protects neonates against infection with an attaching and effacing pathogen. These may also explain that AAMs supplementation could reduce serum ALP, ALT, AST and D-LA levels. The results showed that AAMs supplementation increased immunoglobulin content in colostrum, thereby improving the intestinal barrier function and liver function of piglets.