Effects and mechanisms of nicotinamide supplementation during the peripartum period on glucose and lipid metabolism in lambs from dairy goats

Abstract

This invention discloses the effects and mechanisms of nicotinamide supplementation during the peripartum period on glucose and lipid metabolism in lambs from dairy goats. Starting with maternal nicotinamide supplementation, the study investigated its effects and mechanisms on lamb intestinal morphological development, nutrient transporter expression, glucose metabolism, blood biochemical indicators, and lipid metabolism. The study found that peripartum nicotinamide supplementation in dairy goats can improve glucose absorption in the lamb intestine, inhibit hepatic gluconeogenesis, promote hepatic lipolysis, and reduce liver damage. It can also promote abdominal fat deposition and intestinal morphological development in lambs, thus promoting lamb growth and development. The research results provide a theoretical basis for regulating lamb growth and development.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition, specifically relating to the effect of adding nicotinamide to dairy goats during the peripartum period on the glucose and lipid metabolism of offspring lambs. Background Technology

[0002] The perinatal period is a special physiological period for dairy animals, including late pregnancy and early lactation, generally referring to 21 days before and 21 days after parturition. During pregnancy and peak lactation, the metabolism of tissues and organs such as the placenta, mammary glands, fat, muscle, liver, and gastrointestinal tract undergoes significant changes to supply glucose and amino acids for nutrient distribution between mother and fetus and for fetal development. It also provides free fatty acids (FFAs) such as acetic acid, propionic acid, and butyric acid to the liver and mammary glands for gluconeogenesis and fat synthesis to meet the energy and fat requirements of lactation. Changes in fetal nutrition and endocrine status can cause permanent and heritable changes in the metabolism, structure, and physiology of offspring, and these changes can even lead to diseases in offspring (Metcoff et al. 1980). The environmental and nutritional effects on the fetus and newborn can even affect adult reproductive performance. In the six weeks before parturition, the fetus gains weight rapidly, and the mother is the fetus's sole source of nutrition. Therefore, ensuring adequate maternal nutrition in late pregnancy is crucial for fetal development. Studies have shown that supplementing ewes with vitamin A one month before farrowing promotes lamb survival and production performance (Eldaim et al. 2015); supplementing ewes with vitamin E during pregnancy reduces stillbirth rate (Donnem et al. 2015); and supplementing ewes with feed during late pregnancy and early lactation increases lamb birth weight and weight at 60 days after birth (Idris et al. 2010). Therefore, maternal nutrition during the perinatal and early lactation periods is crucial for fetal and early postnatal growth and development. Ensuring adequate maternal nutrition during these periods can promote offspring growth and development and improve production performance.

[0003] Niacin and nicotinamide are two forms of vitamin B3, and they can be interconverted in the body. Nicotinamide is nicotinamide adenine dinucleotide (NAD). + ) and nicotinamide adenine dinucleotide phosphate (NADP) + Nicotinic acid is one of the main precursors of nicotinic acid. Nicotinamide is mainly derived from animals, but nicotinic acid in grains mainly exists in a bound form, resulting in low utilization efficiency. The main sources of nicotinic acid in the rumen of ruminants are: (1) nicotinic acid in feed; (2) nicotinic acid synthesized by rumen microorganisms; (3) tryptophan is converted into nicotinic acid in the liver via the kynurenic acid pathway, but the conversion efficiency is low, and since tryptophan is an essential amino acid, this part accounts for a small proportion.

[0004] Current research on nicotinamide's effects on glucose metabolism includes: niacin supplementation in peripartum dairy cows significantly reduces the expression of glucose transporter 2 (GLUT2) in the liver (Kinoshita et al. 2016); using obese and diabetic mice as models, nicotinamide supplementation regulates glucose metabolism and the NAD-sirtuin pathway and may be related to alterations in mitochondrial function (Yang et al. 2013); niacin supplementation in peripartum dairy cows alters the expression of liver glucose metabolism genes, possibly related to FoxO1 (Asako Kinoshita et al. 2015). Research on nicotinamide's effects on lipid metabolism includes: nicotinamide, as a functional micronutrient, is crucial for energy metabolism; long-term nicotinamide treatment of MSC cells promotes adipogenesis (Shapiro et al. 2016). Liu et al. (2009) found that nicotinamide can control lipolysis and energy balance in bovine fat precursor cells; nicotinamide promotes hepatocyte energy metabolism within a certain concentration range (Zang Kun et al. 2009; Cheng Haijian et al. 2013) and improves lipid metabolism in stressed dairy cows (Sun Xianzhi et al. 2015). Niacin is a good lipid-lowering drug at pharmacological doses, which can reduce low-density lipoprotein cholesterol (LDL) levels and increase high-density lipoprotein cholesterol (HDL) levels. Supplementing Holstein dairy cows with restricted feed by niacin reduces serum non-esterified fatty acid (NEFA) levels (Pires et al. 2007); supplementing peripartum dairy cows with 12 g / d rumen-protective niacin inhibits lipolysis, reduces milk energy output, and improves peripartum energy balance, but has no effect on feed intake (Yuan et al. 2012); supplementing with 24 g / d niacin can reduce blood NEFA levels and inhibit lipolysis in late peripartum dairy cows (Morey et al. 2011); supplementing Chinese crossbred fattening cattle with 1000 mg / kg niacin increases serum HDL levels, reduces LDL, TG, NEFA, total cholesterol (TC), and glycated serum protein levels, and improves meat quality (Yang et al. 2016). All of the above indicate that both niacinamide and niacin can regulate lipid metabolism.

[0005] Studies on nicotinamide in intestinal development include: nicotinamide can regulate the intestinal mucosal barrier and protect intestinal health; HNF-α and PPARα are major intestinal transcription factors that regulate intestinal integrity and function; claudin-1, claudin-5, and ZO-1 are all intestinal tight junction proteins; nicotinic acid upregulates the expression of these factors, indicating that nicotinic acid can enhance the intestinal barrier (Wei et al. 2015); nicotinic acid deficiency can reduce intestinal mucosal immunity and intestinal physiological function (Feng et al. 2016). Therefore, nicotinamide plays a crucial role in intestinal immunity and glucose and lipid metabolism.

[0006] In summary, nicotinamide can influence glucose and lipid metabolism and promote intestinal development. However, research on the effects of maternal nicotinamide supplementation on the glucose and lipid metabolism of offspring is currently lacking. Lamb rearing is a crucial step in dairy goat farming, and laying a good foundation during the lambing stage is essential for the growth, development, and productivity of adult dairy goats. Therefore, studying the effects and mechanisms of maternal nicotinamide on the glucose and lipid metabolism of offspring lambs can provide a theoretical basis for the efficient and healthy growth and development of lambs. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of existing technologies, this invention starts with the addition of nicotinamide to the mother to explore the effects and mechanisms of maternal nutritional intervention with nicotinamide on the glucose and lipid metabolism and intestinal morphology development of lambs, providing a theoretical basis for regulating lamb growth and development.

[0008] This invention is achieved through the following technical solution:

[0009] One of the objectives of this invention is to provide the application of nicotinamide supplementation in perinatal dairy goats in promoting intestinal morphological development and improving the digestion and absorption of nutrients in the lamb's intestines.

[0010] The application aims to increase the height of duodenal villi, the ratio of duodenal villi height to crypt depth (V / C), the ratio of jejunal villi height to crypt depth (V / C), the height of ileal villi, and the ratio of ileal villi height to crypt depth (V / C), thereby promoting the morphological development of lambs and improving their intestinal digestion and absorption capacity.

[0011] The application aims to promote the expression of glucose transporters GLUT2 and SGLT1 in the jejunum of lambs, as well as the expression of GLUT2 in the ileum, thereby promoting the transport and absorption of glucose in the lamb's intestines.

[0012] Another object of the present invention is to provide the application of nicotinamide supplementation in perinatal dairy goats in improving liver function and reducing liver damage in lambs.

[0013] Another object of the present invention is to provide the application of nicotinamide supplementation in perinatal dairy goats in reducing the incidence of fatty liver in lambs.

[0014] The application is to promote the breakdown of fat in lamb liver and inhibit the synthesis of triglycerides.

[0015] Another object of the present invention is to provide the application of nicotinamide supplementation in perinatal dairy goats to promote abdominal fat accumulation in lambs.

[0016] The application aims to promote the synthesis of fatty acids and triglycerides in the abdominal adipose tissue of lambs, thereby enhancing fat synthesis metabolism without weakening fat decomposition metabolism.

[0017] Another object of the present invention is to provide a method for feeding nicotinamide to dairy goats during the peripartum period, by directly administering nicotinamide orally or by adding nicotinamide to the diet.

[0018] Preferably, the method involves feeding the animal once in the morning and once in the evening, with each feeding being 2.5g.

[0019] Beneficial effects of the present invention

[0020] This invention discloses for the first time that adding nicotinamide to dairy goats during the perinatal period can promote the development of the small intestine morphology of lambs, improve the digestion and absorption of nutrients in the lamb intestine, improve the liver function of lambs and reduce liver damage, reduce the occurrence of fatty liver in lambs and promote abdominal fat deposition in lambs. The related mechanisms are studied, providing a theoretical basis for regulating the growth and development of lambs through maternal nutrition. Attached Figure Description

[0021] Figure 1 Effects of nicotinamide supplementation during the peripartum period on the expression of glucose transporter in the lamb intestine of dairy goats.

[0022] Figure 2 Effects of nicotinamide supplementation during the peripartum period on the expression of gluconeogenesis-related enzyme genes in the liver of lambs in dairy goats.

[0023] Figure 3 Effects of nicotinamide supplementation during the peripartum period on liver glycogen content and gene expression of key metabolic enzymes in lambs from dairy goats.

[0024] Figure 4 Effects of nicotinamide supplementation during the peripartum period on the levels of SIRT1, PGC1α, FoxO1, and SREBP1 mRNA in the liver of lambs in dairy goats.

[0025] Figure 5 Effects of nicotinamide supplementation during the peripartum period on the relative expression levels of ACC, FAS, and SCD mRNA in lamb livers of dairy goats.

[0026] Figure 6 Effects of nicotinamide supplementation during the peripartum period on the relative expression levels of ACC, FAS, and SCD mRNA in abdominal fat of lambs in dairy goats.

[0027] Figure 7 Effects of nicotinamide supplementation during the peripartum period on the relative expression levels of key enzymes in triglyceride metabolism in lamb livers in dairy goats.

[0028] Figure 8 Effects of nicotinamide supplementation during the peripartum period on the relative mRNA expression levels of key enzymes in abdominal triglyceride metabolism in lambs in dairy goats.

[0029] Figure 9 Effects of nicotinamide supplementation during the peripartum period on the relative expression levels of key enzymes in fatty acid oxidation and transport in lamb livers of dairy goats.

[0030] Figure 10 Effects of nicotinamide supplementation during the peripartum period on PGC1α and SREBP1 mRNA levels in abdominal fat of lambs in dairy goats. Detailed Implementation

[0031] The following examples further illustrate the features and other related characteristics of the present invention in detail, to facilitate understanding by those skilled in the art:

[0032] Experiment location: Animal experiments were conducted at the Animal Husbandry Experiment Base of Northwest A&F University.

[0033] Experimental animals: Fifteen dairy goats with two parities were selected for the experiment and paired according to weight and feed intake, divided into three groups: a control group (C group), a group supplemented with nicotinamide 1 to 28 days postpartum (P group), and a group supplemented with nicotinamide 21 days before farrowing to 28 days postpartum (EP group). Nicotinamide was administered twice daily, morning and evening, at a dose of 2.5g each time. After lambing, the lambs in the corresponding groups were L... C Group, L P Group and L EP Five lambs from each group were artificially fed colostrum within half an hour of birth. The colostrum from each group was first mixed thoroughly and then filtered. The milk sample was heated to approximately 74°C, then cooled to approximately 37°C before being fed to the lambs at 05:00, 10:00, 15:00, and 20:00. (The following examples all follow this feeding method.)

[0034] The primers used in this invention were synthesized by Xi'an Qingke Zexi Biotechnology Co., Ltd.

[0035] Example 1: Effects of nicotinamide supplementation during the peripartum period on intestinal morphology development and nutrient transporter expression in lambs from dairy goats.

[0036] 1. Sample collection

[0037] 1.1 Intestinal Segment Collection: Lambs were slaughtered at 28 days of age. The abdominal cavity was quickly opened, and the duodenum, jejunum, and ileum were separated. Intestinal segments of about 3 cm were cut from the middle of each segment. The intestinal contents were rinsed with physiological saline and fixed in 4% paraformaldehyde solution. The segments were then stored in a 4°C refrigerator for intestinal morphology analysis.

[0038] 1.2 Intestinal mucosa collection: Lambs were slaughtered at 28 days of age. The abdominal cavity was quickly opened, and the duodenum, jejunum and ileum were removed. The intestinal contents were rinsed with physiological saline and then the intestinal segments were cut longitudinally and laid flat. The intestinal mucosa was then gently scraped off with a glass slide, aliquoted into cryovials, and quickly placed in liquid nitrogen for short-term storage. Then, it was transferred to a -80°C freezer for storage until analysis.

[0039] 2. Intestinal morphology determination

[0040] The intestinal segments were removed from the fixative and rinsed with water overnight. Following an increasing concentration, the intestinal tissue was dehydrated in ethanol, cleared and permeated with paraffin using xylene, and then embedded in a paraffin-filled mold. After cooling, the tissue was sectioned. Paraffin sections underwent dewaxing, staining (hematoxylin-eosin HE staining), clearing, and mounting with neutral resin. The sections were then observed, and typical and representative fields of view were selected to measure the villus height and crypt depth of the duodenum, jejunum, and ileum. The ratio of villus height to crypt depth was then calculated.

[0041] 3. The expression levels of relevant genes in various intestinal mucosa were detected by RT-PCR.

[0042] GAPDH was selected as an internal reference gene. Primers for SGLT1 and GLUT2 were designed using Primer Primier 5.0 software. RT-PCR was performed on RNA extracted from tissues to detect the expression levels of related genes in various intestinal mucosa.

[0043] 4. Results

[0044] 4.1 Effects of nicotinamide supplementation during the peripartum period on the morphological development of the small intestine in lambs in dairy goats

[0045] Table 1. Effects of nicotinamide supplementation during the peripartum period on the small intestinal morphology of lambs in dairy goats.

[0046]

[0047] Note: Different lowercase letters in the same row subscript indicate significant differences (P<0.05), different uppercase letters in the subscript indicate extremely significant differences (P<0.01), and the same or no subscript indicates no significant differences (P>0.05); n=5; the same applies below.

[0048] As shown in Table 1, L P and L EPThe height of the duodenal villi in group L was higher than that in group L. C Group (P<0.05), L EP The height of the ileal villi in group L is higher than that in group L. C and L P There were no significant differences in jejunal villus height among the groups (P<0.05). There were also no significant differences in crypt depth in the duodenum, jejunum, and ileum among the groups (P>0.05). P Group and L EP The duodenal V / C ratio of the group was higher than that of the L group. C Group (P<0.01), L P Group and L EP The jejunal V / C ratio of the group was higher than that of the L group. C Group (P<0.05), L EP The ileum V / C ratio was higher than that of the L group. C and L P Group (P<0.01).

[0049] Intestinal mucosal morphology, especially the villus and crypt structures, is an important reference indicator for assessing the digestive and absorptive capacity of the small intestine and can also reflect intestinal health (Albrecht et al. 2007). Increased villus height indicates a larger surface area for nutrient absorption (Caspary et al. 1992), while shorter villus and deeper crypts lead to less nutrient absorption, thus reducing production performance (Xu et al. 2003). Increased villus height and the villus height-to-crypt depth ratio are associated with promoting epithelial cell turnover (Fan et al. 1997), and longer villus height can effectively activate cell mitosis (Samanya et al. 2002). In this invention, late maternal supplementation with nicotinamide increased the villus height in the duodenum and the villus height-to-crypt depth ratio in the jejunum of lambs, while maternal supplementation with nicotinamide throughout the maternal period increased the villus height in the ileum and the villus height-to-crypt depth ratio. This indicates that maternal supplementation with nicotinamide can promote the morphological development of the lamb's small intestine and improve the digestive and absorptive capacity of the lamb's intestine.

[0050] 4.2 Effects of nicotinamide supplementation during the peripartum period on the expression of glucose transporter in the lamb intestine of dairy goats

[0051] like Figure 1 As shown, there were no significant differences in the relative expression levels of GLUT2 and SGLT1 in the lamb duodenum among the groups (P>0.05). P The relative expression level of GLUT2 in the jejunum of lambs from group A was higher than that of L. C Group and L EP Group (P<0.01), L C Group and L EP There were no significant differences between groups (P>0.05). P Group and L EPThe relative expression level of SGLT1 in the jejunum of group A lambs was higher than that in group L. C Group (P<0.01), L P Group and L EP There were no significant differences between groups (P>0.05). EP The relative expression level of GLUT2 in the ileum of lambs from group A was higher than that of L. C and L P Group (P<0.05), L C Group and L P There were no significant differences between groups (P>0.05). P Group and L EP The relative expression level of SGLT1 in the ileum of lambs in group L was lower than that in group L C Group (P<0.01), L P Group and L EP There were no significant differences between the groups (P>0.05).

[0052] After lambs consume their mother's milk, the milk coagulates in the abomasum. This coagulation breaks down, and the proteins and fats enter the duodenum for digestion. The final products of digestion are primarily glucose, galactose, lactose, amino acids, and small peptides. These products are then absorbed and utilized by the small intestine to provide nutrition and promote the lamb's growth and development. According to the classic model of hexose absorption in the small intestine, glucose and galactose are transported across the intestinal epithelial cell membrane using the Na-dependent SGLT1 transporter. However, the transfer from cells to the bloodstream requires the hexose-facilitated diffusion transporter GLUT2. SGLT1 and GLUT2 play a synergistic role in the process of glucose crossing the basolateral membrane of intestinal cells into the circulation (Kellett et al. 2008). Therefore, GLUT2 and SGLT1 play crucial roles in intestinal glucose transport and absorption. (et al. 2014). This invention detected the expression levels of SGLT1 and GLUT2 in the duodenum, jejunum, and ileum. The results showed that maternal nicotinamide supplementation did not significantly affect the expression of SGLT1 and GLUT2 in the lamb duodenum; late-stage maternal nicotinamide supplementation significantly increased the expression of SGLT1 and GLUT2 in the lamb jejunum; and maternal nicotinamide supplementation throughout the entire maternal period significantly increased the expression of SGLT1 in the lamb jejunum and GLUT2 in the lamb ileum, but had no significant effect on the expression of GLUT2 in the jejunum. Therefore, this experiment shows that maternal nicotinamide supplementation significantly improves glucose transport and absorption in the lamb jejunum.

[0053] Example 2: Effects of nicotinamide supplementation during the peripartum period on blood biochemical parameters and glucose metabolism in lambs from dairy goats.

[0054] 1. Collect blood samples

[0055] Blood was collected from the jugular vein of lambs at 14 and 28 days of age, 3 hours after morning feeding, using vacuum blood collection tubes. A portion of the blood was centrifuged at 4°C and 3500 rpm for 15 minutes to obtain plasma; another portion was placed at 37°C for 30 minutes and then centrifuged at 3500 rpm for 15 minutes to obtain serum. Both serum and plasma were stored at -80°C for analysis.

[0056] 2. Tissue sample collection

[0057] Lambs were slaughtered at 28 days old. The abdominal cavity was quickly opened, liver tissue was collected, washed with physiological saline, and then stored in liquid nitrogen.

[0058] 3. Measurement Indicators and Methods

[0059] 3.1 Effects of nicotinamide supplementation during the peripartum period on lamb plasma parameters in dairy goats

[0060] Table 2. Effects of nicotinamide supplementation during the peripartum period on plasma parameters in lambs from dairy goats.

[0061]

[0062] Blood biochemical indicators GOT and GPT are important indicators of liver health. GOT and GPT can promote the transfer of α-amino acids from aspartic acid and alanine to the α-keto acid of ketoglutarate, producing oxaloacetate and pyruvate, respectively, which enter the tricarboxylic acid cycle. In this invention, maternal supplementation with nicotinamide throughout the entire pregnancy showed a trend of reducing blood GOT levels in 28-day-old lambs. The nicotinamide-treated group showed a significant reduction in blood GPT levels in 28-day-old lambs, indicating that maternal supplementation with nicotinamide can improve lamb liver function and reduce liver damage.

[0063] 3.2 Effects of nicotinamide supplementation during the peripartum period on serum hormone levels in lambs from dairy goats

[0064] As shown in Table 3, there were no significant differences among the groups in serum insulin, glucagon, leptin, IGF1, IGF2, and insulin sensitivity index in lambs (P>0.05). Therefore, in this invention, the addition of nicotinamide during the peripartum period in dairy goats did not have a significant effect on the serum insulin and glucagon levels in lambs.

[0065] Table 3. Effects of nicotinamide supplementation during the peripartum period on serum hormone levels in lambs from dairy goats.

[0066]

[0067] 3.3 Effects of nicotinamide supplementation during the peripartum period on the expression of key gluconeogenesis enzymes in lamb livers of dairy goats

[0068] Depend on Figure 2 It can be seen that L P and L EPThe relative expression level of PEPCK in group L was significantly lower than that in group L. C Group (P<0.01), L EP The relative expression level of PEPCK in group L was significantly lower than that in group L. P The relative expression levels of G6P, FBP, and PC in lamb livers did not differ significantly among the groups (P>0.05).

[0069] 3.4 Effects of nicotinamide supplementation during the peripartum period on liver glycogen content and expression of key enzymes in liver glycogen metabolism in lambs from dairy goats

[0070] Depend on Figure 3 It can be seen that L EP The liver glycogen content of the group of lambs was lower than that of L C and L P Group trend (P<0.10). L P Group and L EP The relative expression levels of GS and GLUT2 in the livers of lambs in group A were significantly lower than those in group B. C Group (P<0.05), L P Group and L EP There were no significant differences between groups (P>0.05). The relative expression levels of PYGL in lambs did not differ significantly among groups (P>0.05).

[0071] 3.5 Effects of nicotinamide supplementation during the peripartum period on the relative expression levels of SIRT1, PGC1α, FoxO1, and SRBEP1 in the liver of lambs from dairy goats

[0072] Depend on Figure 4 It was found that the relative expression levels of PGC1α and FoxO1 in the livers of lambs in the LP and LEP groups were significantly lower than those in the LC group (P<0.05), while there was no significant difference between the LP and LEP groups (P>0.05). The relative expression levels of SIRT1 and SREBP1 did not differ significantly among the groups (P>0.05).

[0073] In summary, this invention showed that the addition of nicotinamide during the peripartum period of dairy goats did not significantly affect the serum insulin and glucagon levels of lambs. Protein urinary ... Nicotinamide increases blood glucose levels, decreases muscle glycogen content, and induces oxidative stress (Shi et al. 2009). This differs from the blood glucose results in this experiment, possibly because the animals in this experiment were in a normal physiological state, which is inconsistent with the mechanism by which nicotinamide exerts its effects under pathological conditions.

[0074] In this invention, maternal supplementation with nicotinamide throughout the entire pregnancy tends to reduce liver glycogen content in lambs. Liver glycogen content depends on the balance between glycogen synthesis and breakdown. To clarify the reason for the decrease in lamb liver glycogen, this invention investigated the quantitative expression levels of GS and PYGL. Quantitative results for GS showed that the expression of GS in the liver of lambs in the group receiving maternal supplementation with nicotinamide throughout the entire pregnancy was significantly reduced, but had no significant effect on PYGL. Therefore, the possible reason for the decrease in liver glycogen is that maternal supplementation with nicotinamide inhibits FoxO1 expression in the lamb liver, reduces GS gene expression, and decreases liver glycogen synthesis, leading to a decrease in liver glycogen content.

[0075] Gluconeogenesis is regulated by PC, PEPCK, FBP, and G6P. This experiment analyzed changes in the expression of key gluconeogenic enzymes at the transcriptional level to reflect alterations in gluconeogenic capacity. Hepatic gluconeogenesis is crucial for maintaining blood glucose homeostasis. PEPCK is the rate-limiting enzyme in the gluconeogenic pathway. In this invention, maternal nicotinamide supplementation significantly reduced the mRNA expression level of PEPCK in lamb livers, but had no significant effect on FBP, PC, and G6P. GLUT2 in the liver is a bidirectional glucose transporter, ensuring the entry and exit of glucose within hepatocytes. In this experiment, maternal nicotinamide supplementation significantly reduced GLUT2 expression in lamb livers, indicating reduced hepatic glucose output. Therefore, perinatal nicotinamide supplementation in dairy goats may inhibit hepatic gluconeogenesis and reduce hepatic glucose output by suppressing PEPCK expression. SREBP-1C is crucial for glucose-stimulated GLUT2 gene expression; SREBP-1C can activate the GLUT2 promoter region, and GLUT2 is regulated by SREBP-1C (Im et al. 2005). However, in this invention, maternal nicotinamide supplementation did not affect the expression of SREBP-1 in lamb livers; therefore, the altered GLUT2 expression may be due to other factors. In this invention, nicotinamide supplementation during both the entire peripartum period and the postpartum period in dairy goats significantly reduced the expression of PGC1α and FoxO1 in lamb livers. Therefore, peripartum nicotinamide supplementation in dairy goats inhibits the expression of PGC1α and FoxO1 in lamb livers, thereby inhibiting the expression of their target gene PEPCK, suppressing hepatic gluconeogenesis, reducing the amount of glucose transported out of the liver, and decreasing GLUT2 expression. Combined with the previous PUN results, the decreased PUN level and reduced substrate content for hepatic gluconeogenesis may be the main reason for the decreased hepatic gluconeogenesis. Increased intestinal glucose absorption and decreased hepatic gluconeogenesis may explain the lack of significant changes in blood glucose levels in 28-day-old lambs.

[0076] Example 3: Effects and Mechanisms of Nicotinamide Supplementation in Dairy Goats During the Perinatal Period on Lamb Fat Metabolism

[0077] 1. Sample collection

[0078] Lambs were slaughtered at 28 days of age. The abdominal cavity was quickly opened, and abdominal fat tissue and liver tissue were collected. After being washed with physiological saline, the tissues were stored in liquid nitrogen.

[0079] 2. Measurement Indicators and Methods

[0080] 2.1 Measurement of Apparent Indicators of Liver and Abdominal Fat

[0081] Liver tissue and abdominal adipose tissue were removed from a -80℃ freezer, thawed, and then added with appropriate amounts of physiological saline and anhydrous ethanol. The mixture was homogenized and centrifuged at 3000 r / min for 20 min. The supernatant was collected and used for the determination of TG, FFA, TC and abdominal glycerol content.

[0082] 2.2 Enzyme activity assays in liver and peritoneal adipose tissue

[0083] Liver tissue and abdominal adipose tissue were removed from a -80℃ freezer, thawed, and then added with appropriate amounts of pH 7.4 PBS and anhydrous ethanol (adipose tissue has a high fat content, so anhydrous ethanol is necessary for extraction). The mixture was thoroughly mixed and homogenized using a homogenizer. The homogenate was centrifuged at 3000 rpm for 20 min, and the supernatant was collected and used for enzyme activity determination.

[0084] The levels of GPAM, AGPAT6, DGAT2, ATGL, and HSL in liver and peritoneal adipose tissue homogenates were determined using a double-antibody sandwich method.

[0085] 2.3 Measurement of mRNA expression of key enzymes in liver and abdominal fat metabolism

[0086] Primer sequences for AGPAT6, ACC, FAS, SCD, and SREBP1 were referenced from Shi et al. (2013), primer sequences for ATGL were referenced from Lin et al. (2013), primer sequences for DGAT2 were referenced from Bionaz and Loor (2008), primer sequences for GPAM were referenced from Ma and Corl (2012), and primer sequences for HSL were referenced from Li et al. (2015). Primers for CPT1A, ApoB, and MTTP were designed using Primer Primier 5.0 software to determine the mRNA expression of key enzymes in liver and abdominal fat metabolism.

[0087] 3. Results

[0088] 3.1 Effects of nicotinamide supplementation during the peripartum period on FFA, TG, and TC levels in the liver and abdominal fat of lambs.

[0089] Table 4. Effects of nicotinamide supplementation during the peripartum period on FFA, TG, and TC levels in liver and abdominal fat of lambs from dairy goats.

[0090]

[0091]

[0092] As shown in Table 4, L P and L EP The liver FFA content in the group was significantly higher than that in the L group. CGroup (P<0.05), L P and L EP There were no significant differences between groups (P>0.05). EP The FFA content in the abdominal adipose tissue of the group of lambs was significantly higher than that of the group of lambs. C Group (P<0.05). L P and L EP The TG content in the peritoneal adipose tissue of the group was significantly higher than that of the L group. C Group (P<0.01), L EP The TG content in group L was significantly higher than that in group L. P There were no significant differences in liver TG and TC content and peritoneal adipose tissue glycerol and TC content among the groups (P<0.01).

[0093] 3.2 Effects of nicotinamide supplementation during the peripartum period on the expression of key enzymes in de novo fatty acid synthesis in lambs from dairy goats

[0094] Depend on Figure 5 It can be seen that L EP The relative expression level of FAS in the liver of lambs in group A was significantly lower than that in group L. C Group (P<0.05), L C and L P There were no significant differences between groups (P>0.05). EP The relative expression level of ACC in the group and L C The level decreased by 3.52-fold compared to the previous group, but the difference was not significant (P>0.05). There was no significant difference in the relative expression level of SCD among the groups (P>0.05).

[0095] Depend on Figure 6 It can be seen that L EP The relative expression level of FAS in the abdominal fat of lambs in group A was significantly higher than that in group L. C and L P Group (P<0.05), L C and L P There was no significant difference in the relative expression level of FAS among the groups (P>0.05). There were no significant differences in the relative expression levels of ACC and SCD in abdominal fat among the groups (P>0.05).

[0096] 3.3 Effects of nicotinamide supplementation during the peripartum period on the expression of key enzymes in triglyceride metabolism in lambs from dairy goats

[0097] Depend on Figure 7 It was found that there were no significant differences in the relative expression levels of GPAM, AGPAT6, DGAT2, and ATGL in lamb liver among the groups (P>0.05). The relative expression level of HSL in the LP group was significantly lower than that in the LC group (P<0.05), while there was no significant difference between the LP and LEP groups (P>0.05).

[0098] Depend on Figure 8The results showed that the relative expression level of GPAM in the abdominal fat of lambs in the LEP group was significantly higher than that in the LC and LP groups (P<0.01). The relative expression level of AGPAT6 in the LP and LEP groups was significantly higher than that in the LC group (P<0.01), while there was no significant difference in the relative expression level of AGPAT6 between the LP and LEP groups (P>0.05). There was no significant difference in the relative expression level of DGAT2 among the groups (P>0.05). There were no significant differences in the relative expression levels of ATGL and HSL in the abdominal fat of lambs among the groups (P>0.05).

[0099] 3.4 Effects of nicotinamide supplementation during the peripartum period on fatty acid oxidation and transport in lamb liver and abdominal fat transcription factors in dairy goats

[0100] Depend on Figure 9 It can be seen that L EP The relative expression level of ApoB in the liver of lambs in group A was significantly lower than that in group L. C Group (P<0.05), L C Group and L P There were no significant differences between groups (P>0.05). EP The relative expression level of MTTP in the liver of lambs in group A was significantly higher than that in group L. C and L P Group (P<0.01), L C Group and L P There were no significant differences between groups (P>0.05). There were no significant differences in the relative expression levels of CPT1A and ACSL among the groups (P>0.05).

[0101] Depend on Figure 10 It can be seen that L EP The relative expression levels of PGC1α and SREBP1 in the abdominal fat of lambs in group A were significantly higher than those in group B. C and L P Group (P<0.05), L C and L P There were no significant differences between the groups (P>0.05).

[0102] 3.4 Effects of nicotinamide supplementation during the peripartum period on the enzyme activities of key enzymes in liver and abdominal fat metabolism in lambs from dairy goats.

[0103] Table 5. Effects of nicotinamide supplementation during the peripartum period on the enzyme activity of key enzymes in liver lipid metabolism in lambs from dairy goats.

[0104]

[0105] Table 6. Effects of nicotinamide supplementation during the peripartum period on the enzyme activity of key enzymes in abdominal fat metabolism in lambs from dairy goats.

[0106]

[0107] As shown in Table 5, L EPThe AGPAT6 activity in the livers of lambs in the group was significantly lower than that in L C and L P Group (P<0.05), L C and L P There were no significant differences between groups (P>0.05). P HSL activity in group was significantly higher than that in group L C Group (P<0.05), L EP Group and L C There were no significant differences between groups (P>0.05). There were no significant differences in GPAM, DGAT2, and ATGL enzyme activities among the groups (P>0.05).

[0108] As shown in Table 6, there were no significant differences in the enzyme activities of GPAM, AGPAT6, DGAT2, ATGL and HSL in the abdominal fat of lambs among the groups (P>0.05).

[0109] In conclusion,

[0110] 1. Effects and mechanisms of nicotinamide supplementation during the peripartum period on fat metabolism in the abdominal adipose tissue of lambs in dairy goats.

[0111] The addition of nicotinamide during the peripartum period of dairy goats significantly increased the TG content in the abdominal fat of lambs, indicating increased lipid deposition in lambs. Fat deposition depends on the balance between liposynthesis and lipocatabolism. In order to clarify the mechanism of increased TG in the abdominal fat of lambs, this invention detected the expression of genes related to liposynthesis and lipocatabolism.

[0112] The mRNA expression of PGC1α in abdominal fat of lambs supplemented with nicotinamide throughout the perinatal period was significantly increased, and PGC1α can regulate the expression of SREBP1. SREBP1 is an important transcriptional regulator of lipidogenesis, which can selectively activate the expression of key genes in de novo fatty acid synthesis (Osborne et al., 2000). Overexpression of SREBP1-c can increase the expression of lipid synthesis genes, downregulate the expression of lipid oxidation genes, and increase the synthesis and accumulation of TG (Li et al., 2014). In this experiment, the expression of SREBP1 in abdominal fat of lambs supplemented with nicotinamide throughout the perinatal period showed a significant increase, which is consistent with the study by Zhao et al. (2012). Enzymes involved in de novo fatty acid synthesis and TG synthesis are mainly regulated at the transcriptional level (Wang et al., 2015). Therefore, this experiment mainly explores the regulation of SREBP1 expression and its important lipid-producing target genes in offspring abdominal fat by maternal nicotinamide supplementation at the transcriptional level. ACC, FAS, SCD, and GPAM are important target genes of SREBP1 (Shimano et al., 2001). Among them, ACC, FAS, and SCD are three key enzymes in the de novo synthesis of fatty acids. ACC catalyzes the formation of malonyl-CoA from acetyl-CoA, then acetyl-CoA and malonyl-CoA form palmitic acid under the catalysis of FAS, and finally, the carbon chain is elongated under the catalysis of SCD to form long-chain fatty acids. In this experiment, the expression of the FAS gene in the abdominal adipose tissue of lambs supplemented with nicotinamide throughout the perinatal period was significantly increased, indicating that the supplementation of nicotinamide throughout the perinatal period can promote the de novo synthesis of fatty acids in the abdominal adipose tissue of lambs.

[0113] GPAM, AGPAT6, and DGAT2 are important enzymes in the TG synthesis process. Increasing GPAM expression can significantly promote TG synthesis (Yuet et al., 2017). In this invention, the expression of GPAM and AGPAT6 in abdominal fat of lambs supplemented with nicotinamide throughout the peripartum period was significantly increased, and the expression of AGPAT6 in abdominal fat of lambs supplemented with nicotinamide in the late peripartum period was significantly increased. Therefore, it can be concluded that peripartum nicotinamide supplementation in dairy goats can promote TG synthesis in abdominal fat of lambs, thereby enhancing lipid metabolism.

[0114] ATGL and HSL are key enzymes in TG breakdown. Increased expression of ATGL and HSL genes can promote lipolysis (Karbowska et al., 2012; Huang et al., 2017). In this invention, nicotinamide supplementation throughout and after the peripartum period did not significantly affect the expression of ATGL and HSL in lamb abdominal fat, indicating that peripartum nicotinamide supplementation did not significantly affect lamb fat metabolism. The increased fat synthesis metabolism and lack of significant changes in catabolism may be the reason for the increased TG content in abdominal fat.

[0115] 2. Effects and mechanisms of nicotinamide supplementation during the peripartum period on liver fat metabolism in lambs from dairy goats.

[0116] Adding nicotinamide to dairy goats significantly increased the FFA content in lamb livers. To investigate the reasons, this experiment detected the gene expression of lipid synthesis-related genes (SIRT1, PGC1α, SREBP1, ACC, FAS, SCD, GPAM, AGPAT6, DGAT2), and simultaneously detected the gene expression of lipolysis genes (ATGL, HSL), lipid oxidation genes (CPT1A, ACSL), and lipid transport genes (ApoB, MTTP).

[0117] In this invention, maternal supplementation with nicotinamide throughout the entire perinatal period significantly reduced the expression of FAS in lamb liver, while having no significant effect on the expression levels of ACC and SCD. In the later stages of maternal supplementation, nicotinamide significantly increased the expression level of HSL in lamb liver, while the treatment effect had no significant effect on the expression levels of GPAM, AGPAT6, DGAT2, and ATGL. The quantitative results indicate that perinatal nicotinamide supplementation in dairy goats can promote hepatic lipolysis by increasing the mRNA expression level of HSL in lamb liver, but has no significant effect on the mRNA expression of DGAT2. Regarding the activity of key enzymes in lipid metabolism, the results showed that perinatal nicotinamide supplementation in dairy goats significantly reduced the enzyme activity of AGPAT6 in the liver, but had no significant difference in the enzyme activities of GPAM and DGAT2. Combined with the quantitative results, this suggests that nicotinamide supplementation in dairy goats inhibits TG synthesis by inhibiting AGPAT6 activity, but has no effect on mRNA. This may be because the regulation of AGPAT6 in lamb liver by maternal nicotinamide supplementation is mainly at the protein level, rather than the mRNA level. Late-term maternal supplementation with nicotinamide significantly increased HSL enzyme activity. Quantitative results showed that late-term maternal supplementation with nicotinamide promoted hepatic lipolysis by increasing HSL mRNA levels and enzyme activity. The regulation of HSL in lamb liver by maternal nicotinamide supplementation was manifested at both mRNA and protein levels. These results indicate that nicotinamide supplementation in dairy goats can increase hepatic lipolysis and inhibit TG synthesis. Enhanced hepatic TG breakdown may be the reason for increased FFA in lamb liver.

[0118] ApoB and MTTP are crucial for lipoprotein assembly and secretion. MTTP, an intramembrane lipid transporter, promotes the secretion of ApoB-containing lipoproteins, and both are associated with inhibited TG synthesis. This invention shows that nicotinamide supplementation in dairy goats can inhibit TG synthesis in lamb livers. Quantitative results show that maternal supplementation with nicotinamide throughout the entire pregnancy significantly reduced ApoB mRNA expression levels and significantly increased MTTP expression in lamb livers. This indicates that nicotinamide reduces TG synthesis in lamb livers, promotes TG degradation, and reduces ApoB secretion. The increased MTTP mRNA expression level suggests increased secretion of ApoB-containing lipoproteins, which may increase VLDL-TG secretion and potentially lead to elevated plasma TG levels. Perinatal nicotinamide supplementation in dairy goats did not significantly affect the expression of CPT1A and ACSL in lamb livers, indicating that maternal nicotinamide supplementation does not affect fatty acid oxidation in lamb livers.

[0119] The effects of nicotinamide on SIRT1 are debated. Nicotinamide is an inhibitor of SIRT1 (Li et al. 2015; Peledet al. 2012; Shan et al. 2013), but some reports suggest that while nicotinamide is an inhibitor of SIRT1 in vitro, it may be an activator of SIRT1 intracellularly (Hwang et al. 2017). However, in this study, nicotinamide supplementation in dairy goats did not significantly affect the relative expression level of SIRT1 in lamb livers. This may be because the lambs were too young, requiring significantly higher basal NAD levels than older animals (Koltai et al. 2010), or it may be because the amount of nicotinamide entering the lambs in this study was significantly lower than the dose that inhibited SIRT1 activity in cell experiments (Fulco et al. 2008). In this study, nicotinamide supplementation in dairy goats did not affect the expression of either SIRT1 or SREBP1 in lamb livers; therefore, the regulation of fat metabolism in lamb livers by maternal nicotinamide supplementation may be unrelated to SIRT1 and SREBP1.

[0120] PGC1α plays an important regulatory role in systemic and hepatic glucose and lipid metabolism. In this experiment, nicotinamide supplementation in dairy goats reduced the relative expression level of PGC1α in lamb livers, indicating that PGC1α can mediate the process of maternal nicotinamide supplementation regulating lamb lipid metabolism. Maternal nicotinamide supplementation inhibits the expression of PGC1α in lamb livers, thereby inhibiting the mRNA expression of its downstream target gene FAS, the enzyme activity of AGPAT6, increasing the mRNA expression and enzyme activity of HSL, reducing ApoB secretion, inhibiting triglyceride synthesis in lamb livers, and promoting lipolysis.

[0121] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not a limitation on the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. The application of nicotinamide supplementation in peripartum dairy goats in promoting intestinal morphological development and improving intestinal digestion and absorption in lambs, characterized in that, Add nicotinamide to dairy goats during the peripartum period, from 21 days before calving to 28 days after calving; Increase lamb duodenal villus height and the ratio of duodenal villus height to crypt depth (V / C), increase lamb jejunal villus height to crypt depth (V / C), increase lamb ileal villus height and the ratio of ileal villus height to crypt depth (V / C), thereby promoting lamb morphological development and improving lamb intestinal digestive and absorptive capacity. It promotes the expression of glucose transporters GLUT2 and SGLT1 in the jejunum of lambs, and the expression of GLUT2 in the ileum, thereby promoting the transport and absorption of glucose in the lamb intestine.

2. The application according to claim 1, characterized in that, Nicotinamide can be administered orally or added to the diet.

3. The application according to claim 2, characterized in that, Feed once in the morning and once in the evening, 2.5g each time.

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