Sialic acid-porcine milk, pig production and human health

Sialic acids (Sias) are 9 carbon acidic sugar molecule and are key monosaccharide constituents of sialylated glycoproteins (Sia-GP’s), gangliosides and human sialylated milk oligosaccharide (Sia-MOS’s). Human milk sialylated glycoconjugates (Sia-GC’s) are bioactive compounds known to act as prebiotics and promote neurodevelopment, immune function and gut maturation in the newborn. Only limited data are available on the Sia content of porcine milk. In the meantime, the importance of Sia in pig health, especially in the growth, development and immunity of piglet and its overall importance in pig production still remains unknown. On the other hand, due to evolutionary loss of de novo biosynthesis of N- glycolylneuraminic acid (Neu5Gc) in human, Neu5Gc containing animal derived food, particularly red meat is the only source of metabolically incorporated Neu5Gc into human tissue which is currently proposed to be associated with cancer initiation and progression, atherosclerosis and other form of inflammatory diseases. However, modest to no information is available on the Sia, especially Neu5Gc content of different animal species derived red meat including organs. It was hypothesized that, 1. Porcine milk contains significant amount of Sias, where the major form of Sia is N-acetylneuraminic acid (Neu5Ac) and there is a difference in Sia content between sow and gilt (first parity sow) milk. 2. Supplementation of sialylated glycoprotein, bovine milk lactoferrin (bLF) to gilts during gestation and lactation improves gilt and piglet health, growth and development and thereby, improves pig production. 3. Red meat (skeletal muscle and organs) derived glycans from different animal species contain varying amounts of different forms of Sia, Neu5Ac, Neu5Gc and ketodeoxynonulsonic acid (KDN). To address these hypotheses three studies were carried out. In the first study, I quantitatively determined the developmental changes of total level of the Sias, Neu5Ac, Neu5Gc, and KDN in sialylated glycoprotein (Sia-GP’s), sialylated oligosaccharides (Sia-MOS’s) and gangliosides of porcine milk, including also gilt and sow milk during lactation (Jahan et al. J Dairy Sci 2016). The milk from 8 gilts and 22 sows was collected at 3 stages of lactation (colostrum, transition and mature milk). Standard and experimental samples were derivatized with DMB (1,2-diamino-4,5-methylenedioxy-benzene) and analyzed by ultra-high performance liquid chromatography (UHPLC) using a fluorescence detector. The following new findings are reported: (1) Gilt and sow milk contained significant levels of total Sia with the highest concentration in colostrum (1238.5 mg/L) followed by transition milk (778.3 mg/L) and mature milk (347.2 mg/L); (2) During lactation, the majority of Sia was conjugated to Sia-GP’s (41-46%), followed by Sia-MOS’s (31-42%) and a smaller proportion in gangliosides (12-28%); (3) Neu5Ac was the major form of Sia (93-96%) followed by Neu5Gc (3-6%) and then KDN (1-2%), irrespective of the milk fraction and/or stage of lactation; (4) The concentration of Sia in Sia-GP’s and Sia-MOS’s showed a significant decline during lactation, while the level of ganglioside Sia remained relatively constant; (5). Mature gilt milk contained a significantly higher concentration of Sia-GP’s compared to sow milk. The high concentration of total Sia in porcine milk suggests that Sia-GC’s are important nutrients that contribute to the optimization of neurodevelopment, immune function, growth and development in piglets. This also provides an important rationale for the inclusion of Sia-GC’s in pig milk replacers to mimic porcine milk composition for the optimal growth and development of piglets. In the second study, gilts were fed with a commercial pig feed supplemented with lactoferrin (LF), a sialylated iron-binding glycoprotein, which performs multiple beneficial functions including modulating immunity and improves neurodevelopment, health and growth performance. The treatment group was supplemented with 1g LF/day, while the control group was supplemented with 1g milk casein/day from day 1 post mating throughout gestation and lactation. The milk production and body weight gain was monitored. The immunity status of gilts and piglets was measured using ELISA. Our study showed that maternal LF supplementation to the gilt (1) significantly increased milk production at different time points of lactation compared to the control (p<0.001); (2) significantly increased body weight gain of their piglets during the first 19 days of life compared to the control group (p<0.05); (3) tended to increase pregnancy rate, litter size and birth weight, number of piglets born alive, and decrease the number of dead and intrauterine growth restriction (IUGR) piglets; (4) significantly increased the concentration of IgA in gilt serum and sIgA in piglet serum (p<0.05). Our findings suggest that maternal LF intervention in gilts can improve milk production, pig production and immunity and therefore, plays a key role in shaping the performance of their progeny. In the third study I determined the level of free and conjugated forms of Neu5Ac, Neu5Gc and KDN in the fresh skeletal muscle, 5 organs and fat tissue of 9 animal species using ultra-high performance liquid chromatography (UHPLC) to discuss Neu5Gc associated meat quality and human health. Our findings showed that- (1) among all tissues, fat tissue (~32-67µg/g lipid) and skeletal muscle (~207-814 µg/g protein) had the lowest levels of total Sia and lungs had the highest (~2364-5318 µg/g protein) in all species, except dog where kidney contained the highest level of total Sia (~3050 ug/g protein); (2) ~75-98% of total Sia was found in the conjugated form in all organs and species, but dog muscle had more in the free form ( 54%) and cat muscle had similar concentration of conjugated and free Sia (58% and 42 % respectively); (3) Neu5Ac was the major form of Sia in all the organ tissues of 9 animal species except spleen of cattle (~85% Neu5Gc), pig (~75% Neu5Gc), horse (~75% Neu5Gc) and cat (~60% Neu5Gc); (4) Neu5Gc levels in all organs of tested species were ~2–54% higher than that of muscles, except kidney of cattle, goat and horse which had a ~0.3%, ~5% and 8% lower content of Neu5Gc than muscle respectively and heart of horse and kangaroo which had ~5% and ~0.5% lower content of Neu5Gc than muscle respectively. Surprisingly all organs of female deer did not contain any Neu5Gc regardless of whether it was in the free or conjugated form; (5) spleen contained the highest levels of Neu5Gc in the majority of animal species including goat, sheep, horse, cat and kangaroo whereas, in cattle, pig and dog, lung was the highest Neu5Gc containing organ; (6) fat tissue (~ 2-30 µg/g lipid), and muscle (~37-166 µg/g protein) contained the lowest Neu5Gc than any other organ tissues; importantly muscles of kangaroo and dog did not contain Neu5Gc; (7) the majority of Neu5Gc and Neu5Ac in tested organs of species was present in the conjugated form (>70%); (8) <1 % of total Sia was KDN, in which ~ 60%-100% was in the free form, except sheep liver (~75% conjugated) and goat muscles (~88% conjugated). The outcome of this study confirms that Neu5Gc expression is tissues- specific and species-specific. The gender-specificity in deer implies that that estrogen might play a role as direct modulators of cell behaviour in Neu5Gc synthesis and Neu5Gc might play a role in animal production/reproduction. The low levels of Neu5Gc in all skeletal muscle compared to organs, in particular, the absence of Neu5Gc in kangaroo and dog muscle suggests that dietary consumption of muscle meats should be encouraged to reduce red meat associated incidence of cancer, cardiovascular and other inflammatory diseases. Female deer organ meat is a good source of Neu5Ac and micronutrients for human nutrition.