Parental betaine supplementation promotes gosling growth with epigenetic modulation of IGF gene family in the liver

Abstract

Betaine is widely used as a feed additive in the chicken industry to promote laying performance and growth performance, yet it is unknown whether betaine can be used in geese to improve the laying performance of goose breeders and the growth traits of offspring goslings. In this study, laying goose breeders at 39 wk of age were fed basal (Control, CON) or betaine-supplemented diets at low (2.5 g/kg, LBT) or high (5 g/kg, HBT) levels for 7 wk, and the breeder eggs laid in the last week were collected for incubation. Offspring goslings were examined at 35 and 63 d of age. The laying rate tended to be increased (P = 0.065), and the feed efficiency of the breeders was improved by betaine supplementation, while the average daily gain of the offspring goslings was significantly increased (P < 0.05). Concentrations of insulin-like growth factor 2 (IGF-2) in serum and liver were significantly increased in the HBT group (P < 0.05), with age-dependent alterations of serum T3 levels. Concurrently, hepatic mRNA expression of the IGF gene family was significantly increased in goslings derived from betaine-treated breeders (P < 0.05). A higher ratio of proliferating cell nuclear antigen (PCNA)-immunopositive nuclei was found in the liver sections of the HBT group, which was confirmed by significantly upregulated hepatic expression of PCNA mRNA and protein (P < 0.05). Moreover, hepatic expression of thyroxine deiodinase type 1 (Dio1) and thyroid hormone receptor β (TRβ) was also significantly upregulated in goslings of the HBT group (P < 0.05). These changes were associated with significantly higher levels of global DNA 5-mC methylation, together with increased expression of methyl transfer genes (P < 0.05), including betaine-homocysteine methyltransferase (BHMT), glycine N-methyltransferase (GNMT), and DNA (cytosine-5-)-methyltransferase 1 (DNMT1). The promoter regions of IGF-2 genes, as well as the predicted TRβ binding site on the IGF-2 gene, were significantly hypomethylated (P < 0.05). These results indicate that gosling growth can be improved by dietary betaine supplementation in goose breeders via epigenetic modulation of the IGF gene family, especially IGF-2, in the liver.

Parental betaine supplementation improves the growth performance of offspring gosling.

Parental betaine enhances hepatic IGF-2 expression in offspring gosling with DNA hypomethylation on the IGF-2 gene promoter.

Introduction

Betaine, also known as trimethylglycine, is mainly derived from choline oxidation and dietary intake. It can serve as a methyl donor in various methylation reactions in the body (Day and Kempson, 2016). Betaine supplementation in the diet improves egg production in laying hens (Omer et al., 2020; Zaki et al., 2023) or promotes growth in broiler chickens (Chen et al., 2018). Also, betaine has been reported to alleviate the heat stress in laying (Attia et al., 2016) and broiler (Uyanga et al., 2022) chickens and to decrease the abdominal fat deposition in broiler chickens (Leng et al., 2016) and geese (Yang et al., 2021). Furthermore, the transgenerational effects of maternal betaine on offspring performance have been reported in various animal species, including rodents (Zhao et al., 2017; Sun et al., 2023), pigs (Cai et al., 2014; He et al., 2020; Azad et al., 2022), and chickens (Abobaker et al., 2019; Hu et al., 2020).

The goose industry plays important roles in economics, cultures, and ecosystems, yet the low laying and growth rates of many indigenous breeds hinders the development of the goose farming. As herbivorous waterfowl, geese have an advantage over other poultry species in fiber digestion and utilization due to their strong gizzard (Li et al., 2017). Additionally, their gut microbiota can also effectively breakdown the fiber in the cecum and large intestine (Liu et al., 2018; Fang et al., 2023). Furthermore, foodstuffs traverse the gastrointestinal tract more rapidly in geese compared to other poultry species (Zhang et al., 2013). In breeding farms, geese are commonly kept in floor pens with males and females mixed to allow natural mating. Therefore, it is intriguing to investigate whether parental feeding of betaine-supplemented diet affects the laying performance of goose breeders and the growth rate of offspring goslings.

Both insulin-like growth factor (IGF) and thyroid hormone (TH) are crucial regulators of growth and development in poultry (King and May, 1984; McMurtry et al., 1997; Scanes, 2009). The liver is the primary source of endocrine IGFs that exert their biological functions via binding to IGF receptors. Meanwhile, IGF-binding proteins 1-6 (IGFBP1-6) act as carrier proteins in the circulation to regulate the bioavailability and half-life of IGFs (LeRoith et al., 2021). Various factors, including maternal nutritional programming, modulate gene expression in the liver. Maternal intake of methyl donors during pregnancy increases hepatic IGF-1 expression in offspring piglets (Jin et al., 2018). Additionally, hepatic expression of metabolic genes is regulated by maternal betaine supplementation in pigs (Cai et al., 2016) and chickens (Hu et al., 2020). Thyroxine (T4), the major secretory product of the thyroid gland, can be converted into the more bioactive triiodothyronine (T3) through deiodination processes. T3 exerts its action by directly binding to thyroid hormone receptors (TR), which are members of the nuclear receptor superfamily, thereby regulating the transcription of target genes (Ortiga-Carvalho et al., 2016). Plasma T4 levels are reported to increase in post-hatch chicks, while plasma T3 levels increase during early growth stages and decline with age thereafter (McGuinness and Cogburn, 1990; Lu et al., 2007). In chickens, a close relationship between IGFs and THs has been reported that THs affect the production of IGF-1 by stimulating the expression of hepatic growth hormone receptor (GHR) (Tsukada et al., 1998). Conversely, IGF-1 stimulates thyroid cell proliferation and differentiation (Smith, 2021). Nevertheless, the effects of betaine supplementation in parental diet on IGFs and THs in offspring goslings have not been investigated.

Betaine is a substrate in the methionine cycle for its ability to convert homocysteine to methionine and thus participate in epigenetic regulation for gene expression (Day and Kempson, 2016). Previously, we found that maternal betaine supplementation induces intergenerational changes in hepatic IGF-1 gene expression and DNA methylation in F1 and F2 rat offspring (Zhao et al., 2017). Also, maternal betaine treatment causes hypermethylation of differentially methylated regions (DMR) on the IGF-2 gene, resulting in higher IGF-2 gene expression in the hippocampus of newborn piglets (Li et al., 2015). Although IGF-2 is not an imprinting gene in poultry (Nolan et al., 2001; Beatty et al., 2022), in ovo folic acid injection induces hepatic IGF-2 gene overexpression in newly hatched chickens by decreasing DNA methylation in the gene promoter region (Liu et al., 2016). However, whether betaine supplementation in goose breeders affects the hepatic expression of growth-related genes through epigenetic modifications in offspring liver remains unknown.

Therefore, this study aimed to investigate the effects of betaine supplementation on the laying performance of goose breeders and the growth traits of offspring goslings and to explore the possible mechanisms by determining the serum hormones and hepatic expression of genes involved in growth regulations, thyroid hormone metabolism, and methionine metabolism, as well as the status of DNA methylation on the promoter of affected genes in offspring goslings.

Materials and Methods

Ethics statement

All the experiments were approved by the Animal Ethics Committee of Nanjing Agricultural University. The project number is 31972638. The sampling procedures followed the “Guidelines on Ethical Treatment of Experimental Animals” (2006) No. 398 set by the Ministry of Science and Technology, China.

Animals and experimental design

Jiangnan White goose breeders (450 females and 90 males at 39 wk of age) were raised in Changzhou Four Seasons Poultry Farm (Jiangsu Lihua Animal Husbandry Co., Ltd.). Geese were randomly assigned to three groups, each comprising of five replicates. Geese of each replicate were kept in separate floor pens (4 m × 2 m) with 30 females and 6 males per pen mixed to allow natural mating. Geese were fed basal (Control, CON) or betaine-supplemented diets at low (2.5 g/kg, LBT) or high (5 g/kg, HBT) levels for 7 wk (75% purity, Beijing Xin Dayang Co. Ltd.). The ingredient composition and nutrient contents of the experiment diets are shown in Table 1. All geese were reared at 25 to 28 °C, 50% to 60% humidity, and a 16:8 light–dark cycle, with free access to food and water throughout the experiment.

Table 1.

Ingredients and nutrient composition of the basal diet for goose breeders and offspring goslings (as-fed basis)

Items	Goose breeders	1 to 21 d	Gosling diets 22 to 42 d	43 to 63 d
Ingredients, %
Corn	48.27	36.77	37.38	43.78
Wheat	20.00	20.00	20.00	20.00
Rice bran meal	4.00	4.00	4.00	4.00
Soybean meal1	14.20	28.60	25.90	16.70
Corn gluten meal	3.00	2.00	2.00	3.00
DDGS 2		3.00	4.00	5.00
Soybean oil	1.10	1.20	2.50	3.50
Limestone	7.16	1.50	1.45	1.45
Dicalcium phosphate	0.81	1.49	1.39	1.16
Methionine	0.11	0.24	0.18	0.17
Lysine	0.35	0.20	0.20	0.24
Premix3	1.00	1.00	1.00	1.00
Total	100.00	100.00	100.00	100.00
Calculated nutrient levels, %
Metabolizable energy, MJ/kg	12.33	11.72	12.13	12.55
Crude protein	15.00	20.50	18.50	15.5
Crude fat	3.31	3.36	4.85	6.14
Crude ash	10.63	6.60	6.02	5.42
Calcium	3.00	1.00	0.95	0.90
Total phosphorus	0.57	0.76	0.67	0.60
Digestible lysine	0.70	1.00	0.90	0.75
Digestible total sulfur amino acids	0.55	0.73	0.66	0.56

¹The protein content of the soybean meal is 43%.

²The protein content of DDGS is 27%.

³Premix provided per kilogram of diet: vitamin A, 7,000 IU; vitamin D, 4,000 IU; vitamin E, 20 IU; vitamin K, 1.5 mg; vitamin B1, 2 mg; vitamin B2, 10 mg; vitamin B6, 3 mg; vitamin B12, 0.02 mg; niacin, 50 mg; pantothenic acid, 10 mg; folic acid, 1 mg; biotin, 0.2 mg; choline, 400 mg; Fe, 60 mg; Cu, 10 mg; Mn, 100 mg; Zn, 90 mg; I, 0.5 mg.

Measurement of the laying performance

During the whole experiment, the feed intake, egg number, egg weight, and unqualified eggs (egg weight < 115 g or > 185 g, sand-shelled egg, misshaped egg, and broken egg) in each replicate were recorded daily. Daily egg production rate, qualified egg rate, and average egg weight were calculated. Feed conversion ratio (FCR) was calculated as the amount of feed consumed required to produce a unit (g) of egg mass (feed conversion ratio = g feed/g egg).

Fertilized eggs laid last week were collected from each group and incubated at a commercial hatchery. One-day-old goslings from each group were manually sexed and reared in brood cages from 1 to 21 d and transferred to separate floor pens from 22 to 63 d. Each group comprised 5 replicates with 32 goslings each (half male and half female). The goslings were reared under the temperature maintained at 30 to 32 °C for the first week and then reduced approximately 1 °C every 2 d until 21 °C. The relative humidity was kept at 30 % to 50%. All goslings had free access to water and diet (Table 1). At 35 and 63 d, two geese per replicate were randomly selected and sacrificed by rapid decapitation according to the American Veterinary Medical Association Guidelines for Animal Euthanasia: 2013 edition. Blood samples were collected from the wing vein, and serum was separated and stored at −20 °C. Liver samples were stored at −80 °C until use.

ELISA for IGF-1 and IGF-2 concentrations in serum and liver

Serum IGF-1 (YB-IGF1-Go) and IGF-2 (YB-IGF2-Go) levels were measured using commercial ELISA kits and purchased from Shanghai Yubo Biological Technology Co., Ltd., China, following the manufacturer’s instructions. The intra- and inter-assay coefficients of variations of all the kits were 9% and 11%, respectively.

Radioimmunoassay for serum concentrations of thyroid hormones

Serum T4, T3, and FT3 were measured using radioimmunoassay kits purchased from Beijing North Institute of Biological Technology Co., Ltd., China, following the manufacturer’s instructions. The intra- and inter-assay coefficients of variations of all the kits were 10% and 15%, respectively.

Histological and immunohistochemistry analysis

Liver tissues were fixed in 4% paraformaldehyde and then embedded in paraffin blocks. Five-micrometer slices were stained with hematoxylin and eosin (H&E). HE-stained sections were imaged with a light microscope (Olympus-BX53, Tokyo, Japan). The immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in gosling liver tissues was conducted by Wuhan Powerful Biology Co., Ltd. The images were acquired under a light microscope (Olympus-BX53, Tokyo, Japan). PCNA-positive staining cells were counted by using an Image-Plus 6.0 system. The results are expressed as the ratio of positive staining cells to total cell number.

RNA isolation and real-time PCR for mRNA quantification

Total RNA was isolated from 30 mg liver samples using TRIzol reagent (TSP401, Tsingke Biotech Co., Ltd, Nanjing, China), and 1 µg of RNA was reverse transcribed to cDNA according to the manufacturer’s protocol (RK20429, ABclonal Technology Co., Wuhan, China). Diluted cDNA (1:20, vol/vol) was used for real-time PCR, performed with QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Peptidylprolyl Isomerase A (PPIA) was chosen as an internal control. Data were analyzed using the method of 2^−ΔΔCT. All the primers (Supplementary Table S1) were synthesized by Tsingke Biotechnology Co., Ltd. (Nanjing, China).

Total protein extraction and western blotting

Total protein was extracted from 50 mg frozen liver samples and homogenized with RIPA buffer added with protease inhibitor cocktail (B14001, Selleckchem, USA). According to the manufacturer’s instructions, protein concentrations were measured using the BCA Protein Assay Kit (23225, TransGen, Beijing, China). Western blot analysis of PCNA (BS5842, Bioworld, China, diluted 1:1,000), thyroid hormone receptor β (TRβ, A1582, Abclonal, China, diluted 1:1,000), thyroxine deiodinase type 1 (Dio1, 11790-1-AP, Proteintech, Chicago, diluted 1:1,000), DNA (cytosine-5-)-methyltransferase (DNMT1, 24206-1-AP, Proteintech, Chicago, IL, diluted 1:1,000), betaine-homocysteine methyltransferase (BHMT, 15965-1-AP, Proteintech, Chicago, IL, diluted 1:1,000), glycine N-methyltransferase (GNMT, 18790-1-AP, Proteintech, Chicago, diluted 1:1,000), and DNA methyltransferase 3A (DNMT3A, A16834, Abclonal, China, diluted 1:1,000) was carried out following the manufacturer’s instructions. β-Actin (AC026, ABclonal, China, diluted 1:100,000) was used as internal control.

Methylated DNA dot blotting

Hepatic DNA was extracted from the CON and HBT groups. Two hundred and fifty nanogram per microliter DNA sample was denatured at 95 °C for 10 min and transferred onto a hybond membrane (GE Healthcare, Piscataway, NJ, USA). After UV cross-linking, the membrane was blocked with 5% milk for 1 h and incubated with an anti-5mC antibody (ab10805, Abcam, diluted 1:1,000) overnight at 4 °C. The next day, the membrane was washed three times with TBST, followed by incubation with a secondary antibody at room temperature for 2 h. An imaging system (Bio-Rad, USA) was used to capture the pictures, and the dot density was analyzed with ImageJ software, with the staining of 0.02% methylene blue as loading control.

Methylated DNA immunoprecipitation analysis

High-quality genomic DNA was isolated from liver samples (CON and HBT groups) and sonicated to produce small fragments about 300 to 500 bp. One microgram of fragmented DNA was heat-denatured at 95 °C for 10 min to produce single-stranded DNA and a portion of the denatured DNA was stored as input DNA. The immunoprecipitation was performed by using the 5mC antibody overnight at 4 °C (ab10805, Abcam, diluted 1:1,000), and the immune complexes were captured by pretreated protein A/G agarose (sc-2003, Santa Cruz Biotechnology, Inc., CA, USA). Finally, the purified DNA was used to amplify the proximal promoter sequence of the affected genes by real-time PCR with specific primers shown in Supplementary Table S1. The putative THR binding sites in IGF-2 promoter were predicted using hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget/#!/prediction). Data were normalized against the input and presented as the fold change relative to the average value of the CON group.

Statistical analysis

A replicate was designated as the experimental unit for performance parameters, whereas other measured indices were based on individual goose. Data were analyzed by one-way ANOVA using IBM SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). The differences among groups were examined by the Tukey test, which were considered significant at P < 0.05. Student’s t-test was used for two parametric groups. Data are presented as means ± SEM.

Results

Laying performance

Compared with the CON group, the laying rate in the LBT and HBT groups was increased by 17.57% and 12.66% (P = 0.065), respectively, and egg mass of goose breeders was increased by 11.88% and 8.83% (P = 0.059). While ADFI, average egg weight, and qualified egg rate were not affected with the betaine supplementation, yet FCR in the LBT and HBT groups was dropped by 10.71% and 7.86% than the CON group (P = 0.065) (Table 2).

Table 2.

Effects of dietary betaine on laying performance of goose breeders

Items4	CON1	LBT2	HBT3	SEM5	P-value
Laying rate, %	30.96	36.40	34.88	1.012	0.065
Egg mass (g/bird/d)	45.52	50.93	49.54	0.987	0.059
ADFI (g/bird/d)	249.15	248.51	248.29	0.222	0.277
FCR (feed/egg, g/g)	5.60	5.00	5.16	0.111	0.065
Average egg weight, g	142.49	141.92	142.02	0.659	0.938
Qualified egg rate, %	87.36	89.60	89.82	0.551	0.128

¹CON, control received only the basal diet.

²LBT, basal diet supplemental with 2.5 g/kg betaine.

³HBT, basal diet supplemental with 5 g/kg betaine.

⁴ADFI, average daily feed intake; FCR, feed conversion ratio.

⁵SEM, standard error of the mean (n = 5).

Growth performance

Compared with the CON group, body weight in the LBT and HBT groups was increased by 10.08%, 11.05% at 21 d (P < 0.05), and by 4.13%, 6.75% at 63 d (P < 0.05), respectively. The ADFI in the LBT and HBT groups was increased by 5.92%, 7.55% than the CON group during 1 to 21 d (P < 0.05). The ADG in the LBT and HBT groups was increased by 11.07%, 12.25% than the CON group during 1 to 21 d (P < 0.05). Meanwhile, FCR in the LBT and HBT groups was dropped by 4.30%, 3.76% than the CON group during 1 to 22 d (P < 0.05). For the whole feeding period from 1 to 63 d, ADG in the HBT group was increased by 7.61% than the CON group (P < 0.05) (Table 3).

Table 3.

Effects of parental betaine supplementation on the growth performance of goslings

Performance measure4	CON1	LBT2	HBT3	SEM5	P—value
1 d BW, g	86.62	87.86	86.68	0.575	0.644
21 d BW, g	1032.79b	1136.93a	1146.90a	16.043	0.002
42 d BW, g	2480.22	2587.63	2625.11	28.764	0.094
63 d BW, g	3663.62b	3814.79a	3911.05a	55.756	0.032
1 to 21 d
ADFI, g	83.93b	88.90a	90.27a	1.000	0.011
ADG, g	44.98b	49.96a	50.49a	0.767	0.002
FCR, g/g	1.86a	1.78b	1.79b	0.018	0.040
22 to 42 d
ADFI, g	215.43	223.78	217.22	2.906	0.501
ADG, g	68.99	69.10	70.39	1.105	0.865
FCR, g/g	3.13	3.24	3.11	0.058	0.637
43 to 63 d
ADFI, g	244.55	247.95	249.52	5.293	0.936
ADG, g	56.35	58.44	61.24	1.955	0.627
FCR, g/g	4.37	4.26	4.13	0.086	0.507
1 to 63 d
ADFI, g	181.30	186.88	185.67	1.932	0.498
ADG, g	56.78b	59.16ab	61.10a	0.886	0.041
FCR, g/g Mortality, %	3.19 8.13	3.16 8.75	3.06 8.12	0.029 2.12	0.179 0.973

^a,bMeans within a row lacking common superscript differ (P < 0.05).

¹CON, control received only the basal diet.

²LBT, basal diet supplemental with 2.5 g/kg betaine.

³HBT, basal diet supplemental with 5 g/kg betaine.

⁴BW, body weight; ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion ratio.

⁵SEM, standard error of the mean (n = 5).

Hormones concentration in serum and liver of goslings

Serum levels of thyroid hormones showed age-dependent alterations in betaine-supplemented groups. At 35 d, compared with the CON group, T4 (Figure 1A) and FT3 (Figure 1C) were increased by 33.26% (P < 0.05), 25.61% (P < 0.05), respectively, in the LBT group, while T3 (Figure 1B) was increased by 55.56% (P < 0.05) in the HBT group. At 63 d, however, no significant differences were observed in T4 (Figure 1A), while serum T3 (Figure 1B) and FT3 (Figure 1C) in the HBT group were dropped by 26.10% (P < 0.05), 25.06% (P < 0.05) than the CON group. As a result, the T3/T4 ratio in the HBT group was dropped by 30.00% than the CON group at 63 d (P < 0.05; Figure 1D).

Figure 1.

Effect of parental betaine supplementation on hormones concentration in serum and the liver of gosling with different ages. (A) Serum T4 level; (B) serum T3 level; (C) serum FT3 level; (D) serum T3/T4 ratio; (E) serum IGF-1 level; (F) serum IGF-2 level; (G) hepatic IGF-1 content; (H) hepatic IGF-2 content. Values are presented as mean ± SEM (n = 10). Bars marked with different superscripts are significant differences (P < 0.05).

Serum IGF-1 (Figure 1E) in the HBT group was increased by 41.78% than the CON group at 35 d (P < 0.05), while serum IGF-2 (Figure 1F) in the HBT group was increased by 19.16% and 10.18% than the CON group at both 35 and 63 d (P < 0.05). Meanwhile, hepatic IGF-1 concentration (Figure 1G) in the HBT group was increased by 43.20% than the CON group at 35 d (P = 0.06). Compared with CON group, the hepatic IGF-2 concentration in the HBT group (Figure 1H) was increased by 84.91% at 35 d (P < 0.05) and by 36.16% at 63 d (P < 0.05).

Phenotypic characterization of histomorphology and proliferation in the liver of goslings

No pathological changes were observed in H&E-stained sections of the gosling liver tissues at 35 d (Figure 2A) or 63 d (Figure 2C). The HBT group had heavier liver weight at both 35 d (P = 0.072) and 63 d (P < 0.05), as compared with the CON group (Figure 2B). The heavier liver in the HBT group appears proportional to the higher body weight, as no alterations were seen in the liver index (Figure 2D). Accordingly, higher number of PCNA-positive cells was observed in the liver of the HBT group at both 35 d (P < 0.05) (Figure 2E, 2F) and 63 d (P < 0.05) (Figure 2G, 2H), which was supported by significantly increased hepatic expression of PCNA at both mRNA (P < 0.05) (Figure 2I) and protein levels (P < 0.05) (Figure 2J).

Figure 2.

Effect of parental betaine supplementation on characterization of histomorphology and proliferation in liver of gosling with different ages. (A) HE staining (200×) of liver tissue at 35 d; (B) liver weight; (C) HE staining (200×) of liver tissue at 63 d; (D) Liver index; (E) immunohistochemical staining (200×) for PCNA in the liver tissue at 35 d; (F) statistical PCNA positive cells ration (%) in the liver tissue at 35 d ; (G) immunohistochemical staining (200×) for PCNA in the liver tissue at 63 d; (H) statistical PCNA positive cells ration (%) in the liver tissue at 63 d; (I) the mRNA expression level of PCNA gene in the liver; (J) the protein expression of PCNA in the liver. Values are presented as mean ± SEM (n = 10). Bars marked with different superscripts are significant differences (P < 0.05).

Expression of hepatic growth-related genes

At 35 d, the mRNA expression of hepatic IGF-2 and IGF-1R (Figure 3A) in both LBT and HBT groups was significantly upregulated (P < 0.05), while at 63 d, the upregulation of hepatic IGF-2, IGF-1R, and GHR mRNA expression (Figure 3B) was observed only in HBT group (P < 0.05). In addition, the mRNA expression of IGFBP1 in the liver of the HBT group was significantly elevated at both 35 d (Figure 3C) and 63 d (P < 0.05) (Figure 3D). Hepatic IGFBP3 mRNA expression showed a significant upregulation in the HBT group at 63 d (P < 0.05) (Figure 3D).

Figure 3.

Effect of parental betaine supplementation on the expression of IGF gene family in liver of gosling with different ages. (A) The mRNA expression level of growth-related genes in the liver at 35 d; (B) The mRNA expression level of IGF-binding proteins in the liver at 35 d; (C) The mRNA expression level of growth-related genes in the liver at 63 d; (D) The mRNA expression level of IGF-binding proteins in the liver at 63 d. Values are presented as mean ± SEM (n = 10). Bars marked with different superscripts are significant differences (P < 0.05).

Expression of hepatic THs metabolism-related genes

At 35 d, Dio1 (Figure 4A) and TRβ (Figure 4B) mRNA expression was significantly increased in the liver of the HBT group (P < 0.05), with the same pattern of increase at protein level (Figure 4C). At 63 d, Dio1 mRNA in the HBT group showed a significant decrease (Figure 4D), while TRβ protein expression was consistently higher in the liver of the HBT group (P < 0.05) (Figure 4F).

Figure 4.

Effect of parental betaine supplementation on the expression of thyroid hormone related gene in the liver of gosling with different ages. (A) The mRNA expression level of Dio1 and Dio3 genes at 35 d, n = 10; (B) The mRNA expression level of TRα and TRβ genes at 35 d, n = 10; (C) Protein expression of Dio1 and TRβ at 35 d, n = 10. (D) The mRNA expression level of Dio1 and Dio3 genes at 63 d, n = 10; (E) The mRNA expression level of TRα and TRβ genes at 63 d, n = 10; (F) Protein expression of Dio1 and TRβ at 63 d, n = 8. Values are presented as mean ± SEM. Bars marked with different superscripts are significant differences (P < 0.05).

Hepatic protein content of methionine cycle and methyl transfer genes

The global DNA 5mC methylation levels were significantly enhanced in the liver of HBT goslings at both 35 and 63 d (P < 0.05) (Figure 5A). DNA methyltransferase (DNMT1) mRNA at both 35 d (Figure 5B) and 63 d (Figure 5C) was significantly increased in the liver of HBT group (P < 0.05), together with significantly increased mRNA expression of betaine-homocysteine (BHMT) and S-adenosyl homocysteine hydrolase-like protein 1 (AHCYL1) at 63 d (P < 0.05). At the level of protein, BHMT, glycine N-methyltransferase (GNMT), and DNMT1 were all significantly upregulated in the HBT group at both 35 d (P < 0.05) (Figure 5D) and 63 d (P < 0.05) (Figure 5E).

Figure 5.

Effect of parental betaine supplementation on the expression of methionine metabolic genes in the liver of gosling with different ages. (A) Global DNA 5-mC level in the liver at 35 and 63 d was detected by dot blot, n = 8; (B) the mRNA expression level of methionine metabolic genes at 35 d, n = 10; (C) the mRNA expression level of methionine metabolic genes at 63 d, n = 10; (D) protein expression of BHMT, GNMT, DNMT1, and DNMT3A at 35 d, n = 8; (E) protein expression of BHMT, GNMT, DNMT1, and DNMT3A at 63 d, n = 8. Values are presented as mean ± SEM. *P < 0.05 and bars marked with different superscripts are significant differences (P < 0.05).

MeDIP analysis for DNA methylation status on the promoter of affected genes

The methylation status of the promoter and coding regions for the IGF-2 gene at 35 d (Figure 6A) and 63 d (Figure 6B) was detected with the MeDIP-PCR technique. The fragment 1 of the IGF-2 gene promoter and the exon 3 of the IGF-2 gene were significantly hypomethylated at 35 d (P < 0.05; Figure 6A). The hypomethylation status on these sites was maintained at 63 d (Figure 6B), with fragment 2 of the IGF-2 gene promoter also being significantly hypomethylated (P < 0.05).

Figure 6.

Effect of parental betaine supplementation on DNA methylation on the promoter and exons of affected genes in gosling liver at different ages. (A) Methylation status on the promoter and exons of IGF-2 at 35 d; (B) methylation status on the promoter and exons of IGF-2 at 63 d; (C) methylation status of TRα and TRβ binding site on the promoter region of IGF-2 at 35 d; (D) methylation status of TRα and TRβ binding site on the promoter region of IGF-2 at 63 d. Values are presented as mean ± SEM (n = 6). *P < 0.05.

Binding sites for both TRα (-519 to -386) and TRβ (-372 to -270) were predicted on the promoter of the IGF-2 gene. MeDIP analyses revealed significant hypomethylation of the TRβ binding site (P < 0.05) in the liver of HBT goslings at 35 d (Figure 6C) and 63 d (Figure 6D).

Discussion

In this study, betaine supplementation tended to enhance the laying production of laying geese, which is consistent with the studies in laying hens that betaine-supplemented diet increases laying performance (Attia et al., 2016; Omer et al., 2020). In addition, parental betaine supplementation exhibited a growth-promoting effect on offspring goslings with increased body weight and improved FCR. Previous studies reported that sows fed betaine-supplemented diet during pregnancy produce heavier piglets at birth, weaning (Jia et al., 2015), and finishing (Jin et al., 2018) stages. Nevertheless, in earlier studies, we reported that maternal betaine did not increase the body weight of the offspring chickens (Hou et al., 2018; Hu et al., 2020). Many factors contribute to inconsistent outcomes, including different types and levels of methyl donors, species, breeds, and the supplementation period. It is noted that geese are kept in floor pens with males and females mixed to allow natural mating in this experiment. Thus, we could not exclude the possibility of paternal effects on the growth performance of the offspring goslings.

The liver is crucial in regulating growth and metabolism and is the main organ for synthesizing and secreting IGF-1 and IGF-2 (Livingstone, 2013). It is also the target organ of IGFs and regulates liver cell proliferation through the IGF-1R signaling pathway (Pivonello et al., 2014). We previously reported that grandmaternal betaine supplementation enhances hepatic IGF-2 expression in F2 rat offspring (Yang et al., 2020). In this study, the expression of proliferation marker gene PCNA was significantly increased in the liver of betaine-treated offspring goslings, which indicates higher cell proliferation in the liver. At 35 d, PCNA was increased at mRNA level, but remained unchanged at protein level. The discrepancy of mRNA abundance and protein content implies the possible involvement of post-transcriptional and translational regulations. Concurrently, the hepatic IGF-2 expression was significantly up-regulated in the betaine group at 35 and 63 d, thereby elevating serum circulating IGF-2. Similar to the previous study, maternal betaine supplementation significantly enhanced the hepatic expression of the IGF-2 gene in offspring rats (Yang et al., 2018). It appears that increased liver cell proliferation in the betaine group might relate to augmented IGF-2 expression and to meet the demands of rapid growth and development of offspring goslings.

In addition, we observed that the serum T3 levels of offspring goslings showed a completely inverse pattern between 35 and 63 d. In previous studies, this phenomenon reported that serum T3 levels were significantly decreased in fast-growing broilers (meat-type) compared with slow-growing broilers (Gonzales et al., 1999; Vaccaro et al., 2022). Thus, the change in thyroid hormone status may affect the body’s metabolic rate, alter nutrient partitioning, and affect meat yield, contributing to more efficient feed utilization and faster growth rate (Vaccaro et al., 2022).

We previously reported that betaine supplementation could affect hepatic IGFs (Yang et al., 2020) and metabolic-related gene expression (Hu et al., 2020) in offspring by modulating carbon metabolism and DNA methylation. Maternal betaine supplementation enhanced the protein expression of BHMT and DNMT1 in the liver of offspring chickens (Hu et al., 2020). Consistent with previous research, the hepatic protein content of BHMT, GNMT, and DNMT1 were markedly increased in the betaine group compared with the control, which demonstrated that the hepatic methionine cycle pathway of offspring goslings was activated through parental betaine supplementation. DNA methylation is one of the primary mechanisms for betaine exerts gene epigenetic regulation (Li et al., 2022). For instance, maternal betaine supplementation significantly increased the hepatic IGF-2 and IGF-1 gene expression in rat offspring, which contributed to hypermethylation status in the imprinting control region of the IGF2 gene (Yang et al., 2018) and hypomethylation in promoter regions of the IGF-1 gene (Zhao et al., 2017).

The effects of betaine on epigenetic gene regulation have been reported in cell (Omer et al., 2020) or animal models (Wang et al., 2013; Li et al., 2015, 2022; Zhao et al., 2020). For instance, maternal betaine, either supplemented in the diet of hens (Abobaker et al., 2019) or injected into the fertilized eggs (Hu et al., 2017), modulates DNA methylation status and thus the gene expression pattern of newly hatched chickens. To validate the similar effects of betaine in geese, we conducted an in ovo injection trial by injecting betaine at 7.5 mg/egg to fertilized goose eggs before incubation (Data are not shown). The dosage of betaine injected was determined based on the range of betaine concentrations in chicken eggs (Zeisel et al., 2003) as well as insights from our previous publications (Hu et al., 2017; Abobaker et al., 2022), while considering the larger size of goose eggs, which are approximately three times the size of chicken eggs. Newly hatched goslings (1-d old) were sacrificed for determining the methylation levels of the hepatic IGF-2 gene promoter. In ovo injection of betaine significantly increased the level of total DNA 5mC methylation in the liver, which was in line with higher mRNA and protein levels of betaine metabolic enzymes. However, the methylation levels of the hepatic IGF-2 gene promoter were significantly decreased, in accordance with what was found at 35 and 63 d of age. Taken together, these results imply that betaine induced hypomethylation of the IGF-2 gene promoter at hatching and maintained to 35 and 63 d of age later in life.

In the somatotropic axis, GH binds to the GHR on the surface of the cell membrane of the target organ, thereby initiating the intracellular responses and promoting the expression of IGFs. The hypermethylation in the promoter region of the hepatic GHR gene in broilers disrupts its interaction with GH and further downregulates the production of IGF-1 (Cong et al., 2023). Normal growth requires the interaction between the thyrotropic axis and the somatotropic axis. For instance, thyroid hormones regulate the expression and release of growth hormone (GH) from the pituitary gland, stimulating the liver to produce IGF-1 (Kühn et al., 2002; Smith 2021). Meanwhile, the growth rate and IGF-1 production were reduced in hypothyroid chickens (Decuypere et al., 1987). As mentioned above, the IGF-2 is also crucial for the growth of poultry. However, no studies report the effects of thyroid hormones on IGF-2 in vertebrate studies. Only one study in the salmon hepatocytes showed that IGF-2 gene level was inhibited by T3 treatment (Pierce et al., 2010). In this experiment, we found that the TRβ binding site on the IGF-2 gene promoter region was hypomethylation, which may facilitate TRβ binding to its specific response element. Thus, we speculated that TH may regulate the transcriptional activation of the IGF-2 gene. Nevertheless, this assumption needs to use the ChIP method or in vitro models to verify whether such a link exists in the betaine treatment.

Conclusion

Taken together, we demonstrate that parental betaine supplementation improved the growth performance of offspring goslings with epigenetic regulation of the IGF-2 gene in the liver at both 35 and 63 d. More in-depth studies are required to elucidate whether other regulatory mechanisms are involved in parental-betaine-induced growth and metabolic regulation in offspring goslings.

Supplementary Data

Supplementary data are available at Journal of Animal Science online.

Glossary

Abbreviations

AHCYL1

adenosylhomocysteinase-like 1

BHMT

betaine-homocysteine methyltransferase

DNMT1

DNA (cytosine-5-)-methyltransferase 1

DNMT3A

DNA (cytosine-5-)-methyltransferase 3A

Dio1

type 1 iodothyronine deiodinase

Dio3

type 3 iodothyronine deiodinase

GHR

growth hormone receptor

GNMT

glycine N-methyltransferase

IGF-1

insulin-like growth factor 1

IGF-2

insulin-like growth factor 2

IGF-1R

insulin-like growth factor 1 receptor

IGFBPs

insulin-like growth factor-binding protein

MAT2B

methionine adenosyltransferase 2B

PCNA

proliferating cell nuclear antigen

PPIA

peptidylprolyl isomerase A

TRβ

thyroid hormone receptor β

TRα

thyroid hormone receptor α

Conflict of Interest Statement

The authors declare that they have no competing interests.

Author Agreement

The corresponding author and all the authors have read and approved the final submitted manuscript. We further confirm that the order of authors listed in the manuscript has been approved by all of us.