Maternal Dietary Citrulline Supplementation Increases Fetal Growth and Programs Pancreatic Development in the Lambs

doi:10.21203/rs.3.rs-7474212/v1

Intrauterine growth restriction (IUGR), caused by maternal undernutrition, impairs fetal growth and increases the risk for postnatal metabolic dysfunction. L-arginine can mitigate these effects; however, its use in sheep is limited by ruminal microbial degradation. Interestingly, L-citrulline, the precursor for arginine synthesis, bypasses ruminal catabolism and may be a practical alternative. This study evaluated if maternal L-citrulline supplementation to nutrient restricted ewes from gestational days (GD) 28 to 140 (term = 147) enhances fetal growth in lambs. Gestating ewes were fed 50% of National Research Council (NRC) nutritional recommendations to induce IUGR and received either L-citrulline (0.40% of diet) or an isonitrogenous alanine control (0.61% of diet). Birth weight and pre-suckling blood samples were collected, and lambs remained with dams until postnatal day 60 (PND60) (citrulline: n = 13; alanine: n = 10). Lambs from L-citrulline treated ewes were heavier at birth (P = 0.05) and PND60 (P < 0.05), with greater (P < 0.05) absolute weights of the pancreas, brain, liver, and small intestine. Pancreatic mass per gram of body weight was greater (P < 0.05) in citrulline lambs. The relative proportion of endocrine and exocrine pancreas were not different between treatments. Circulating insulin concentrations were greater (P = 0.05) at birth and circulating glucose concentrations were increased (P < 0.05) in the citrulline lambs on PND60. These results suggest that maternal L-citrulline supplementation is a viable alternative to arginine for improving fetal growth during maternal malnutrition, with benefits persisting through weaning.

IUGR

lamb

L-citrulline

nutrient restricted

pancreas

Intrauterine growth restriction (IUGR), defined as impaired growth of the mammalian fetus, is a major cause of perinatal morbidity and mortality in mammals (Kesavan et al. 2019; Pilliod et al. 2012, Piper et al. 1996; Resnik et al. 2002; Wu et al. 2006). IUGR can arise from a multitude of factors including placental insufficiency, maternal undernutrition, overnutrition in adolescent pregnancy, and increased fecundity, among other things (Wu et al. 2006). In ruminant livestock production systems, undernutrition is particularly common due to seasonal forage variability and drought (Thomas et al. 1995; Scocco et al. 2016). Beyond birth, IUGR has lifelong consequences, predisposing offspring to metabolic disorders in postnatal life due to persistent developmental programming which occurs in utero (Galjaard et al. 2013; Lumey et al. 1998; Phillips et al. 1994; Resnik et al. 2002). Nutrient restriction during gestation causes permanent structural and functional changes to multiple organs and tissues, including the pancreas, liver, skeletal muscle, and adipose tissue within the fetus (Martin-Gronert et al. 2007).

Undernutrition of gestating females leads to permanent structural and functional changes to multiple organs, particularly the endocrine pancreas (Martin-Gronert et al. 2007). In sheep, the window between gestational days 33 and 147 is critical for isletogenesis and endocrine cell proliferation. Nutrient restriction during this period can result in impaired pancreatic development (Green et al. 2010). IUGR during this window predisposes the fetus to pancreatic dysfunction later in life, characterized by lower circulating insulin and glucose concentrations, small and less populated islets, fewer β-cells, and an overall decrease in pancreatic weight and insulin content in sheep and rats (Boehmer et al. 2017; Dumortier et al. 2007; Ford et al. 2007; Fowden and Hill 2001; Garofano et al. 1999; Green et al. 2010; Limesand et al. 2013; Mohan et al. 2018; Simmons et al. 2007; Zhang et al. 2016b). During pancreas organogenesis, a lack of adequate nutrition can activate endocrine pathways and gene expression regulating mechanisms predisposing the fetus to reduced insulin secretion and an increase in insulin resistance later in life (Armengaud et al. 2021).

L-arginine, a conditionally essential amino acid for the mother and fetus, is reduced in maternal and fetal circulation during times of undernourishment (Bourdon et al. 2020; Wu et al. 2006). Studies have demonstrated that intravenous administration of L-arginine to undernourished gestating females increased the availability of nutrients to support fetal growth and increased birth weight (Lassala et al. 2010; Lin et al. 2014; Zhang et al. 2016a). Yet another study found that L-arginine supplementation to nutrient-restricted ewes from gestational day 100 to 125 did not increase fetal weight but did increase pancreatic mass and brown adipose tissue mass, specifically (Satterfield et al. 2013). L-arginine supplementation to improve fetal growth parameters has shown promising results; however, its application in ruminant livestock production systems is limited due to the extensive catabolism that L-arginine undergoes in the rumen. Overcoming this catabolism requires costly encapsulation for L-arginine to reach the small intestine and be absorbed into the body for utilization (Lassala et al. 2009; Gilbreath et al. 2020). In recent studies, L-citrulline, a precursor of L-arginine, was shown to bypass catabolism by ruminal microbes, thus increasing plasma concentrations of arginine after oral supplementation (Gilbreath et al. 2020). The half-life of L-citrulline in the maternal plasma of pregnant sheep is 1.49 h, which is much longer than that for L-arginine (0.76 h) (Wu 2022). Thus, intravenous administration of L-citrulline to pregnant ewes is more effective than L-arginine for increasing arginine availability in both the mother and the fetus (Lassala et al. 2009).

The objective of this research project was to investigate if dietary supplementation of L-citrulline to ewes from gestational days (GD) 28 to 140 enhances growth and development of the lamb to weaning. Further, we aim to elucidate the effects of dietary L-citrulline supplementation on development and function of the pancreas. We hypothesize that maternal L-citrulline supplementation to nutrient restricted ewes would increase lamb weights at the time of birth, program major metabolic organs such as the pancreas, and in response improve rates of postnatal growth to weaning.

All experimental procedures in this study were approved by the Institutional Animal Care and Use Committee of Texas A&M University (AUP 2021 − 0251).

Animal Study and Tissue Collection

Multiparous Rambouillet donor ewes of similar frame size were subjected to superovulation followed by embryo collection and transfer to generate pregnancies, as described previously (Edwards et al. 2020). A sum of 332.5 IU of follicle stimulating hormone (Follitropin, Vetoquinol; Fort Worth, TX) was administered in decreasing dosages, twice daily, from days 9–12 post insertion of a controlled intravaginal drug-releasing device (CIDR, Eazi Breed CIDR; Zoetis Animal Health, Troy Hills, NJ). Donor ewes were inseminated using fresh semen to a single Hampshire sire, to control for effect of sire on birthweight; by laparoscopic artificial insemination 42 h post CIDR removal. On day 6, Rambouillet ewes were flushed, and a single grade one blastocyst was transferred into the uterus of Hampshire recipient ewes. Recipient ewes of similar body condition were synchronized using a 12-day CIDR protocol. All pregnancies were generated within a one-week period to minimize the impact of climate on the study. Beginning on day 21 of gestation, ewes were individually housed on concrete flooring and fed 100% National Research Council (NRC) requirements divided into two equal feedings at 0700 and 1800 h. Composition of the diet was as previously described (Lassala et al. 2010). On Day 28 of gestation, pregnancy was confirmed by ultrasonography and dietary intake was reduced to 50% of NRC requirements to induce fetal growth restriction. Also at this time, ewes were randomly assigned to one of two supplemental groups [alanine (isonitrogenous control); n = 18) or citrulline (n = 17)]. L-citrulline (> 99.8% purity; obtained from boxnutra.com) was fed at a rate of 0.40% of the diet while alanine (> 99.8% purity; obtained from boxnutra.com) was fed at a rate of 0.61% of the diet to provide equivalent levels of nitrogen. This supplemental dose of citrulline, which was 53% of the arginine content (0.76%) in the basal diet (Satterfield et al. 2013), was the same as that for pregnant gilts fed a diet containing 0.70% arginine (Li et al. 2023). Amino acid supplements were provided as a top dress to the daily ration divided equally at each feeding. The ewes consumed all the feed provided daily.

Sheep were weighed weekly and feed and supplement intakes were adjusted based on changes in body weight. Amino acid supplementation ended on gestational day 140, as ewes neared parturition and dietary intake was increased to 75% NRC until lambing. After parturition ewes received 100% NRC. At the time of lambing a blood sample was collected by jugular venipuncture, centrifuged at room temperature for 10 min at 2000 x g, plasma was collected and stored at -20°C, birth weight and other physical parameters (the alanine group, n = 12; the citrulline group, n = 14) were obtained before suckling was encouraged. Lambs were reared with mom until postnatal day (PND) 60, whereas ewes and lambs were weighed weekly after parturition.

On PND 59, lambs were separated from ewes and fasted prior to necropsy on PND60 (alanine n = 6 females, 4 males; citrulline n = 4 females, 9 males). Due to a small amount of death loss between birth and weaning (n) of lambs from both treatment groups decreased slightly at PND60. At the time of necropsy, body weight was recorded, and a blood sample was collected from each lamb. Lambs were humanely euthanized via jugular intravenous administration of phenytoin and pentobarbital (Beuthanaisa-D). Immediately after euthanasia, lamb organs were harvested and weighed, and physical parameters were measured and recorded. Tissue samples were fixed in 4% paraformaldehyde or were minced, snap-frozen, and stored at -80°C for subsequent analyses.

Determination of Circulating Insulin, Glucose, and NEFA Concentration

Concentrations of insulin, glucose and non-esterified fatty acids (NEFAs) were quantified in plasma collected on PNDs 1 and 60 using commercially available kits, according to manufacturer recommendations. All assays were validated for linearity in plasma from sheep. Insulin was measured using an Ovine Insulin Assay Kit (Cat#10-1202-01; Mercodia, Uppsala, Sweden) (Lassala et al. 2010). Glucose was measured using a Glucose Colorimetric Assay Kit (Cat#STA-680; Cell Biolabs INC, San Diego, CA, USA) (Halloran et al. 2021). NEFAs were measured using a NEFA Assay (Wako Diagnostics, Mountain View, CA, USA) (Steinhauser et al. 2020).

Determination of Pancreatic Histoarchitecture

Immunofluorescence analyses were performed on pancreatic tissue as previously described (Seo et al. 2019). For dual immunofluorescence staining, sequential tissue sections of 5 µm were cut from paraffin-embedded alanine lamb (n = 10) and citrulline lamb (n = 13) pancreases at 120-µm intervals for histological and morphometric evaluation. Pancreatic sections were deparaffinized and rehydrated in an alcohol gradient. For each animal, two non-sequential sections with at least 45 µm between them were stained for α-amylase, glucagon, and a dual-staining of insulin and somatostatin, respectively. Each non-sequential section was analyzed by collecting 40 non-overlapping captures per stain. α-amylase and glucagon stained sections were placed in a steamer for 40 min in 10 mM citric acid buffer pH 6.0 and cooled for 20 min. Sections used for dual immunofluorescence staining for localization of insulin and somatostatin were placed in warmed 1X PBS and 0.025g protease and allowed to incubate at 37°C for 8 min. Sections were washed three times in PBS for 5 min and then blocked with 1.5% normal goat serum in 1X PBS for 60 min. Mature pancreatic endocrine hormones and the exocrine pancreas were identified in the postnatal lamb pancreas with guinea pig anti-porcine insulin (Dako, Carpinteria CA, 1:50), previously described by Limesand et al. (2005), mouse anti-porcine glucagon (Sigma-Aldrich, St. Louis, MO, 1:50), adapted from Limesand et al. (2005), rabbit anti-human somatostatin 28 (Abcam, Cambridge UK, 1:500), and rabbit anti-human pancreatic α-amylase (Cell Signaling Technology, Danvers, MA, 1:50). Primary antibodies were diluted in blocking buffer and incubated at 4°C overnight. Negative controls were included where the primary antibody was excluded. After overnight incubation, pancreatic sections were washed three times for 5 min with PBS; immunocomplexes were observed with secondary antibodies conjugated to Alex Fluor 488, Texas Red, Alexa Fluor 647 (Abcam, Cambridge UK) diluted 1:250 in blocking buffer for 60 min at room temperature. The pancreatic sections were washed three times for 5 min each with PBS and mounted with Prolong Gold with DAPI.

Pancreatic Cell Proliferation

Non-sequential pancreatic sections (n = 2 sections/lamb/treatment) were deparaffinized and rehydrated in an alcohol gradient. Antigen retrieval was achieved by placing the slides in a steamer for 40 min in 10 mM citric acid buffer, pH 6.0. Slides were then cooled for 20 min and washed three times in PBS for 5 min. Sections were blocked with 1.5% normal goat serum in PBS for 60 min. Pancreatic sections were dual-stained for proliferating cells and α-amylase to differentiate between the exocrine and endocrine areas. Proliferating cells were identified with PCNA mouse mAb (Cell Signaling Technology, Danvers, MA, 1:250) and the exocrine pancreas was identified using rabbit anti-human pancreatic α-amylase (Cell Signaling Technology, Danvers, MA, 1:50). Primary antibodies were diluted in blocking buffer and incubated at 4°C overnight; negative controls were included where the primary antibody was excluded. After the overnight incubation, pancreatic sections were washed three times for 5 min with PBS; immunocomplexes were observed with secondary antibodies conjugated to Alex Fluor 488, Texas Red, (Abcam, Cambridge UK) diluted 1:250 in blocking buffer for 60 min at room temperature. The pancreatic sections were washed three times for 5 min each with PBS and mounted with Prolong Gold with DAPI.

Cell proliferation analyses were performed with QuPath software (Bankhead et al. 2017). Pancreatic proliferation was classified PCNA positive or negative for 40 fields of view (FOV) per lamb on 2 pancreatic sections separated by at least 45 µm (citrulline n = 13 alanine n = 10). Total nuclei per FOV (n = 40 FOV/lamb) was calculated and nuclei within vasculature was subtracted from total, in order to determine total nuclei in pancreatic tissue. Nuclei within the endocrine pancreas was calculated and subtracted from total nuclei to calculate the number of nuclei within the exocrine pancreas. PCNA⁺ cells were counted in endocrine and exocrine areas separately to determine the percentage of proliferating cells in the respective areas.

Morphometric Analyses

Fluorescent images were visualized and digitally captured using a Nikon Eclipse Ni-E (Nikon Corp). Morphometric analyses were performed with ImageJ software (Schneider et al. 2012). Pancreatic sections of lambs from citrulline and alanine treated ewes (n = 2 sections/lamb) were evaluated for α-amylase, glucagon, and a dual-stain of insulin and somatostatin. α-amylase was used to label acinar cells of the exocrine pancreas, glucagon labeled α-cells in the endocrine pancreas, insulin and somatostatin labeled β- and δ-cells in the endocrine pancreas, respectively. α-amylase⁺, glucagon⁺, insulin⁺, and δ-cell areas were determined for 40 FOV on two pancreatic sections per lamb per stain separated by at least 45 µm. Blank space per FOV was subtracted from the total area per FOV to obtain a new total area of pancreatic tissue and vasculature.

Vasculature percentage was determined by calculating major vasculature area and dividing by the new total area of pancreatic tissue. Once vasculature percentage was calculated, vascular area was subtracted from total area of pancreatic tissue and vasculature to determine area of secretory pancreatic tissue. Endocrine pancreas percentage was determined by calculating the endocrine area and dividing by the area of pancreatic tissue. Exocrine pancreas area was determined by subtracting the area of the endocrine pancreas from the area of pancreatic tissue. This area was then divided by the area of pancreatic tissue to calculate percentage of the exocrine pancreas. Endocrine pancreas mass was calculated by multiplying the percentage of endocrine pancreas area by the mass of the pancreas. Similarly, exocrine pancreas mass was calculated by multiplying the percentage of exocrine pancreas by the pancreas mass.

Insulin⁺ area percentage was determined by calculating the area of insulin-positive cells, including β-cells exhibiting colocalization of somatostatin, divided by the area of the islets. Glucagon⁺ area percentage was determined by calculating the area of glucagon-positive cells and dividing it by the area of the islets. δ-cell area percentage was determined by calculating the area of somatostatin-positive cells, disregarding β-cells exhibiting colocalization of somatostatin, divided by the area of the islets. β-cell (insulin⁺ cells), α-cell (glucagon⁺ cells), δ-cell mass was determined by multiplying the percent-positive cells by the estimated endocrine mass.

Statistical Analyses

Statistical analyses were performed using a 2-way ANOVA procedure in SAS 9.3 (SAS Inst. Inc., Cary, NC). Main effects (treatment and sex) and their interaction were included. If no effect or interaction of sex was present, it was removed from the statistical model. Data were log transformed, when necessary, after observation of residual plots. Significance was set at P < 0.05 and all data are presented as means ± SEM.

Physical Characteristics of Lambs

Lambs born to ewes supplemented with L-citrulline from days 28 to 140 of gestation were heavier (5.5 vs. 4.9 kg ± 0.2; P = 0.05) at birth compared to those born to ewes supplemented with alanine (Table 1). On PND60, lambs born to citrulline treated ewes were 2.3 kg heavier (P < 0.05; Table 1) than alanine controls. Lambs from L-citrulline treated ewes also tended to exhibit an increased (P = 0.08) crown-rump length compared to alanine controls (Table 2). Among groups, male lambs from L-citrulline treated ewes exhibited the largest sternal circumference, exceeding (P = 0.02) that of females from the same treatment group and both sexes from the alanine group (data not shown).

Table 1 Effects of maternal citrulline supplementation on fetal and postnatal growth rate

	Alanine^a (n=6f,4m)^b	Citrulline^a (n=4f,9m)^b	P-value
Birth Weight (kg)	4.9 ± 0.2	5.5 ± 0.2	0.05
Weaning Weight-PND60^c (kg)	17.1 ± 0.8	19.4 ± 0.7	0.04

^aValues are presented as means ± SE.

^bSex: f, female and m, male.

^cPND60: postnatal day 60.

Table 2 Effects of maternal citrulline supplementation on lamb physical parameters and tissue/organ weights on PND60

	Alanine^a (n=6f,4m)^b	Citrulline^a (n=4f,9m)^b	P-value
Brain (g)	83.2 ± 2.1	88.9 ± 1.7	< 0.05
Pituitary (g)	0.295 ± 0.136	0.305 ± 0.011	0.57
Thymus (g)	22.5 ± 2.5	25.9 ± 2.1	0.30
Right Ventricle (g)	20.4 ± 0.8	21.4 ± 0.7	0.35
Septum (g)	19.3 ± 1.2	19.7 ± 1.0	0.79
Lungs (g)	255 ± 9	263 ± 8	0.54
Adrenals (g)	1.40 ± 0.06	1.48 ± 0.05	0.30
Kidneys (g)	70.3 ± 4.0	73.1 ± 3.3	0.61
Liver (g)	244 ± 8	274 ± 7	0.01
Pancreas (g)	12.6 ± 1.2	18.6 ± 1.0	<0.01
Spleen (g)	66.0 ± 6.3	69.3 ± 5.2	0.69
Rumen (g)	155 ± 14	186 ± 12	0.11
Abomasum (g)	68.7 ± 4.3	70.1 ± 3.6	0.81
Small Intestine (g)	455 ± 28	538 ± 23	0.03
Omentum (g)	107 ± 17	127 ± 14	0.39
Kidney & Pelvic Adipose Tissue (g)	187 ± 31	186 ± 25	1.00
Gastrocnemius Muscle (g)	49.1 ± 2.4	55.3 ± 2.0	0.06
Longissimus Dorsi Muscle (g)	222 ± 12	252 ± 10	0.06
Testes (g)	8.3 ± 1.4	10.9 ± 0.8	0.15
Ovaries (g)	1.6 ± 0.2	1.7 ± 0.3	0.92
Crown Rump Length (cm)	82.1 ± 1.8	86.2 ± 1.3	0.08
Front Leg Length (cm)	14.7 ± 0.4	14.8 ± 0.4	0.88

^aValues are presented as means ± SE.

^bSex: f, female and m, male.

Lamb Organ and Tissue Weights

Maternal citrulline supplementation increased (P < 0.05) the weight of several organs in the lamb on PND60, including the brain, liver, pancreas, and small intestine while also tending to increase (P < 0.10) weights of the gastrocnemius and longissimus dorsi muscles (Table 2). Cardiac measurements revealed a treatment by sex interaction, with male lambs from the L-citrulline group having a greater whole heart mass (P = 0.03) and left ventricle mass (P = 0.01) compared to other groups (Table 3). Lambs from citrulline treated ewes had a heavier (P = 0.01) whole hind limb mass than alanine labs (Table 4). Likewise, male lambs from L-citrulline treated ewes exhibited a greater (P < 0.05) whole hind limb mass than L-citrulline treated females (Table 4).

To compare the growth of the individual organ to the lamb’s body weight, the relative mass was calculated (g organ/kg bodyweight). Pancreatic mass was disproportionately greater (P < 0.05) in lambs from citrulline treated ewes compared to controls (Table 4). Across both treatment groups, male lambs had increased soleus muscle mass relative to body weight compared to females (P < 0.05; data not shown).

Table 3 Effects of maternal citrulline supplementation on lamb physical parameters and tissue/organ weights with treatment and sex interactions on PND60

				P-values
	Sex	Alanine^a (n=6f, 3m)^b	Citrulline^a (n=4f, 9m)^b	Trt	Sex	*TrtSex**
Sternal Circumference (cm)	F	60.0 ± 1.8	59.6 ± 1.4	0.03	0.69	0.02
	M	57.0 ± 1.7	63.7 ± 0.9
Heart (g)	F	76.8 ± 3.7	69.7 ± 4.5	0.47	0.93	0.03
	M	66.9 ± 5.2	80.3 ± 3.0
Left Ventricle (g)	F	34.1 ± 1.6	31.9 ± 1.9	0.14	0.86	0.01
	M	29.4 ± 2.3	37.3 ± 1.3
Whole Hind Limb (g)	F	1096 ± 45	1142 ± 55	0.01	0.98	<0.05
	M	987 ± 64	1249 ± 37

^aValues are presented as means ± SE.

^bSex: f, female and m, male.

Table 4 Effects of maternal citrulline supplementation on relative tissue/organ weights (g organ/kg bodyweight) on PND60

Organ (g organ/kg body weight)	Alanine^a (n=6f,4m)^b	Citrulline^a (n=4f,9m)^b	P-value
Brain	5.0 ± 0.2	4.6 ± 0.2	0.23
Pituitary	0.017 ± 0.0007	0.016 ± 0.0006	0.13
Thymus	1.3 ± 0.1	1.3 ± 0.1	0.99
Right Ventricle	1.22 ± 0.04	1.11 ± 0.04	0.08
Septum	1.15 ± 0.06	1.02 ± 0.05	0.08
Lungs	15.3 ± 0.7	13.6 ± 0.6	0.07
Adrenals	0.083 ± 0.005	0.077 ± 0.004	0.38
Kidneys	4.2 ± 0.3	3.8 ± 0.2	0.24
Liver	14.6 ± 0.7	14.2 ± 0.6	0.73
Pancreas	0.77 ± 0.08	0.97 ± 0.07	<0.05
Spleen	3.9 ± 0.3	3.6 ± 0.3	0.52
Small Intestine	27.2 ± 1.8	28.0 ± 1.5	0.72
Rumen	9.1 ± 0.6	9.6 ± 0.5	0.57
Abomasum	4.0 ± 0.2	3.6 ± 0.2	0.18
Omentum	6.2 ± 0.8	6.5 ± 0.7	0.77
Kidney & Pelvic Adipose Tissue	10.8 ± 1.4	9.4 ± 1.2	0.47
Gastrocnemius Muscle	2.9 ± 0.10	2.9 ± 0.1	0.77
Longissimus Dorsi Muscle	13.0 ± 0.3	13.0 ± 0.3	0.92
Testes	0.54 ± 0.06	0.54 ± 0.03	0.98
Ovaries	0.09 ± 0.01	0.09 ± 0.01	0.98

^aValues are presented as means ± SE.

^bSex: f, female and m, male.

Concentrations of Insulin, Glucose, and NEFAs

Prior to suckling, on PND1, lambs from L-citrulline treated ewes had higher (P = 0.05) plasma insulin concentrations compared to lambs from alanine treated ewes (Table 5). No differences (P > 0.1) were observed in glucose or NEFAs concentrations on PND1 (Table 5). By PND60, lambs from the L-citrulline group exhibited a 25% increase (P < 0.05) in plasma glucose concentration compared to alanine controls (Table 6). No differences (P>0.10) were observed in plasma insulin or NEFA concentrations on PND60 between treatments (Table 6).

Table 5 Effects of maternal citrulline supplementation on concentration of insulin, glucose, and NEFAs in lamb plasma at PND1

	Alanine^a (n=6f,4m)^b	Citrulline^a (n=4f,9m)^b	P-value
Insulin (μg/L)	0.095 ± 0.035	0.190 ± 0.030	0.05
Glucose (mg/dL)	45.6 ± 5.6	48.6 ± 4.9	0.69
NEFA (mEq/L)	0.32 ± 0.08	0.27 ± 0.07	0.62

^aValues are presented as means ± SE.

^bSex: f, female and m, male.

Table 6 Effects of maternal citrulline supplementation on concentration of insulin, glucose, and NEFAs in lamb plasma at PND60

	Alanine^a (n=6f,4m)^b	Citrulline^a (n=4f,9m)^b	P-value
Insulin (μg/L)	0.076 ± 0.017	0.113 ± 0.015	0.13
Glucose (mg/dL)	157 ± 13	196 ± 12	<0.05
NEFA (mEq/L)	0.95 ± 0.11	1.00 ± 0.09	0.72

^aValues are presented as means ± SE.

^bSex: f, female and m, male.

Pancreatic Morphometry

Representative immunofluorescence images highlight the exocrine pancreas (stained pink), with clearly delineated exocrine areas (outlined in white), and vasculature (outlined in yellow) (Fig. 1a,b). These images were utilized to calculate percentage areas. Maternal L-citrulline supplementation did not alter the percentage of areas of either the endocrine or exocrine pancreas, while there was a tendency for a decrease (P = 0.09) in vascular area within the pancreas of lambs whose mothers received citrulline (Table 7).We next estimated the mass of the endocrine and exocrine pancreas by multiplying the percentage areas by the mass of the organ and found that mass of both the endocrine and exocrine pancreas was increased (P = 0.03; P < 0.01) in lambs whose mothers received L-citrulline supplementation during gestation (Table 7).

At PND60, α, β, and δ cells types were observed in both L-citrulline and control lamb pancreases (Fig. 2a,b,d,e,g,h). Cells expressing mature endocrine hormones were localized within islets (Fig. 2d,e,g,h). No difference was observed in the percentage area of either glucagon⁺ or insulin⁺cells, although there was a tendency for a decrease (P = 0.07) in δ-cell area (Table 8). Notably, β-cell mass within the endocrine pancreas was greater (P < 0.01) in lambs from L-citrulline treated ewes compared to controls, as was α-cells mass (P < 0.01; Table 8).

Table 7 Effects of maternal citrulline supplementation on pancreatic characteristics

	Alanine^a (n=6f,4m)^b	Citrulline^a (n=4f, 9m)^b	P-value
Endocrine Pancreas, %	6.1 ± 1.0	7.7 ± 0.9	0.29
Exocrine Pancreas, %	93.8 ± 1.1	92.3 ± 1.0	0.33
Vasculature, %	0.042 ± 0.003	0.035 ± 0.003	0.09
Endocrine Pancreas (g)	0.7 ± 0.2	1.4 ± 0.2	0.03
Exocrine Pancreas (g)	11.5 ± 1.1	16.8 ± 1.0	<0.01

^aValues are presented as means ± SE.

^bSex: f, female and m, male.

Table 8 Effects of maternal citrulline supplementation on endocrine pancreas morphometry

	Alanine (n=6f,4m)	Citrulline (n=4f, 9m)	P-value
Insulin-positive area, %	0.68 ± 0.07	0.67 ± 0.06	0.94
Glucagon-positive area, %	0.22 ± 0.04	0.23 ± 0.03	0.79
δ-cell area, %	0.05 ± 0.01	0.03 ± 0.01	0.07
β-cell mass (g)	0.45 ± 0.08	0.89 ± 0.08	<0.01
α-cell mass (g)	0.14 ± 0.03	0.27 ± 0.02	<0.01
δ-cell mass (g)	0.04 ± 0.01	0.04 ± 0.01	0.75

^aValues are presented as means ± SE.

^bSex: f, female and m, male.

Pancreatic Cell Proliferation

Endocrine and exocrine cell proliferation were examined with PCNA, a marker for cell proliferation (Bologna-Molina et al. 2013; Zargar-Shoshtari et al. 2018). Exocrine areas are shown in pink, with endocrine areas outlined in white for better clarification between the two areas, PCNA⁺cells are stained green (Fig. 3a,b). L-citrulline supplementation to gestating ewes showed no difference in the percentage of PCNA⁺ exocrine pancreas cells between treatment groups (0.016 ± 0.003% vs 0.021 ± 0.003%) (Table 9). Furthermore, a tendency for increased (P = 0.07) percentage of PCNA⁺ endocrine pancreas cells was observed in lambs from L-citrulline treated ewes (0.044 ± 0.014%) when compared to from lambs from control ewes (0.012 ± 0.015%) (Table 9).

Table 9 Effects of maternal citrulline supplementation on pancreatic cell proliferation

	Alanine^a (n=6f,4m)^b	Citrulline^a (n=4f, 9m)^b	P-value
PCNA-positive cells exocrine pancreas, %	0.016 ± 0.003	0.021 ± 0.003	0.41
PCNA-positive cells endocrine pancreas, %	0.012 ± 0.015	0.044 ± 0.014	0.07

^aValues are presented as means ± SE.

^bSex: f, female and m, male.

We have previously reported that maternal L-arginine supplementation in undernourished ewes from days 100 to 125 of gestation (term = 147) results in a 32% increase in fetal pancreatic mass at day 125 of gestation (Satterfield et al. 2013). Additionally, L-arginine supplementation from days 60 of gestation to term has been shown to increase birth weights of lambs from nutrient restricted ewes (Lassala et al. 2010). Importantly, L-citrulline supplementation bypasses ruminal degradation and increases L-arginine levels in both maternal and fetal plasma (Gilbreath et al. 2019, 2020; Lassala et al. 2009). Based on these findings, we conducted the present study to determine if L-citrulline supplementation to malnourished ewes would improve fetal growth and pancreatic development. Results of the present study indicate that maternal L-citrulline supplementation from days 28 to 140 of gestation increased birth and weaning weights of lambs by ~ 13% and increased pancreatic weight by 48% while altering the function of the endocrine pancreas. These observations have significant potential impact on improving fetal growth and preventing metabolic disorders that can occur as a result of IUGR.

Among the various intrauterine factors influencing fetal development, maternal nutrition plays a critical role in regulating both placental and fetal growth (Wu et al. 2004). Malnourished ewes commonly experience excessive body fat loss during gestation and often give birth to low birth weight lambs with diminished tissue reserves, increasing neonatal mortality rates (Dwyer 2008). Numerous animal studies have consistently demonstrated that maternal undernutrition during critical periods of gestation results in reduced fetal weights at necropsy or birth weights at parturition (Bhasin et al. 2009; Hoffman et al. 2014; Luther et al. 2009; Satterfield et al. 2013; Wu et al. 2006). These incidences of maternal malnutrition resulting in fetal growth restriction are an economically important issue for livestock producers (Wu et al. 2004, 2006). Direct intravenous infusion of L-arginine has shown beneficial outcomes in increasing arginine concentrations in both maternal and fetal blood in nutrient restricted ewes (Lassala et al. 2009, 2010). Further, nitric oxide and polyamines, metabolites of arginine, play important roles in mitigating fetal growth restriction (Hsu and Tain 2019; Lefevre et al. 2011). The use of an undernourished sheep model reported that administration of sildenafil citrate, a PDE5 inhibitor (Glossmann et al. 1999), increased fetal weight by 14% from both underfed and adequately fed ewes on gestational day 115 (Satterfield et al. 2010). A nutrient restricted sheep model receiving parenteral administration of L-arginine also demonstrated an increase in birth weight (Lassala et al. 2010). Direct infusion of arginine into the fetal femoral vein for 3–4 hours displayed an increase in fetal whole-body protein in an ovine placental insufficiency IUGR model (de Boo et al. 2005), likely by stimulating MTOR cell signaling, and protein synthesis as reported for porcine placental cells (Kong et al. 2012) and skeletal muscle (Yao et al. 2008). Ewes receiving arginine administration for an extended period of time have demonstrated functional changes in the placenta that may be responsible for enhanced nutrient transport from mother to fetus during rapid fetal growth (Kwon et al. 2004). Results of the current study indicate that maternal L-citrulline supplementation to undernourished ewes between gestational days 28 and 140 led to a 13% increase in birth weight and weaning weight. Due to the prolonged supplementation of L-citrulline to gestating ewes this increase in birth weight is indicative of improved nutrient utilization by the fetus and/or increased delivery of substrates necessary for growth by improved placental function. Previous studies using a nutrient restricted ovine model concluded that maternal supplementation of L-arginine increased birth weight (Lassala et al. 2010; Sun et al. 2018; Zhang et al. 2016a) and lambs from nutrient restricted ewes supplemented L-citrulline had increased bodyweight at postnatal day 19 (Kott et al. 2021), further supporting the present study. Alternatively, studies supplementing L-arginine (Gootwine et al. 2020; Piene et al. 2018; Satterfield et al. 2013) or L-citrulline (Kott et al. 2021) to malnourished ewes have observed no differences in birth weights. The contradictory nature of these studies likely results from a variety of factors including maternal dietary intake levels, supplementation levels, fetal number, and timing and/or duration of supplementation. In the United States, lambs are typically sold by the pound at or shortly after weaning, and therefore the improvement in weaning weight by citrulline is a potentially cost-effective tool to increase revenue for producers.

Fetal development under conditions of maternal malnutrition is often characterized by asymmetric growth, where visceral organs such as the liver and pancreas exhibit reduced mass, while the brain is relatively spared due to preferential blood flow (Serpente et al. 2022). It has previously been reported that offspring at 110 days of gestation from nutrient-restricted ewes receiving rumen protected L-arginine (20g/day) had greater organ weights of the heart, liver, pancreas, small intestine, and large intestine (Zhang et al. 2016a). In the present work, maternal L-citrulline supplementation increased the weights of several organs in lambs at PND60, including the brain, liver, pancreas, and small intestine. These findings suggest that L-citrulline supports organ development in a manner that may counteract the typical consequences of IUGR, especially key metabolic organs that are affected by brain sparing. In contrast, a study by Sun et al. (2018) demonstrated that maternal arginine supplementation (20g/day, divided twice daily) to nutrient-restricted, twin-bearing ewes from gestational day 35–110 enhanced fetal growth but had no effect on the weights of brain, pancreas, and liver. Interestingly, the current study found that pancreas weight relative to the growth of the lamb suggests that maternal L-citrulline supplementation directly targets development of the fetal pancreas. We have previously reported that administration of sildenafil citrate increased pancreatic weight when calculated as a proportion of fetal weight (Satterfield et al. 2010). Sildenafil citrate acts by prolonging the vasodilatory effects of NO through the cGMP pathway, thereby increasing placental perfusion and fetal nutrient availability (Glossman et al. 1999). Of note, L-citrulline supplementation as used in the current study, enhances endogenous NO synthesis due to its role as a precursor to L-arginine (Bahri et al. 2012; Gonzalez et al. 2023). Despite differing mechanisms, both interventions ultimately converge on the NO pathway to support placental function and fetal growth. However, unlike L-citrulline and L-arginine, sildenafil citrate does not provide polyamine or protein production, which are critical for cellular proliferation and organogenesis (Hussain et al. 2017; Mastracci et al. 2015; Wu and Morris 1998; Wu et al. 2021). Due to the increase in relative pancreatic weight this suggests that the activation of the NO vasodilatory pathway may be critical in promoting fetal pancreatic development.

Nutritional models of IUGR in sheep often result in offspring exhibiting low birth weight and an altered rate of postnatal growth and reduced efficiency of feed/forage utilization leading to increased adipose deposition (Wu et al. 2006). In the present study, maternal citrulline supplementation promoted a more desirable lean-to-fat ratio due to an increase in whole hind limb mass and increased gastrocnemius and longissimus dorsi muscle masses with similar levels of adipose tissue to controls. The increase in skeletal muscle mass may be indicative of more efficient nutrient uptake and anabolic metabolism due to the larger small intestine (for nutrient digestion and absorption) and liver (for metabolism including ammonia detoxification) or improved levels of protein synthesis (for lean tissue gain). Further, this suggests that lambs from L-citrulline supplemented ewes were able to maximize available nutrients towards muscle growth and development instead of adipose deposits. With lambs from L-citrulline treated ewes presenting a leaner carcass, this could result in an improved carcass yield and greater cutability which both contribute to increased profitability to the livestock sector. Further studies are warranted to determine if this characteristic would persist to slaughter age (6–8 months of age; ~ 63.5 kg).

In sheep, fetal IUGR is associated with decreased pancreatic weight and β-cell percentage and a concomitant reduction in pancreatic insulin content (Green et al. 2010). Interestingly, arginine supplementation to nutrient-restricted ewes from gestational day 35 to 110 increased insulin concentration despite no difference in pancreatic weight when compared to fetuses from nutrient restricted ewes (Sun et al. 2018). In the present study, the analyses of metabolic hormones in plasma collected immediately after parturition and prior to suckling revealed that L-citrulline supplementation increased insulin concentration at PND1, while no differences were observed in glucose concentrations. Elevated insulin levels in neonates are often associated with increased nutrient supply to the fetus, which may have been facilitated by improved placental function via NO-mediated vasodilation and angiogenesis (Wu et al. 2004) from the supplemented citrulline. Due to PND1 plasma samples being collected immediately after parturition but prior to suckling, plasma components reflect placental transfer or fetal response to the intrauterine environment but not the extrauterine environment. Insulin secretion from β-cells has been shown to be sensitive to amino acids during fetal development (Brown et al. 2011). Further, the entry of arginine, a charged amino acid, is able to directly depolarize the β-cell membrane causing the voltage-gated calcium channel to open, stimulating insulin secretion (Brown et al. 2011). The elevation of insulin at PND1 could be due to citrulline increasing the plasma concentrations of arginine (Gilbreath et al. 2020; Lassala et al. 2009), potentially stimulating insulin secretion from β-cells. This increase of insulin could have also programmed skeletal muscle development, as elevated concentrations of insulin have been shown to enhance skeletal muscle protein synthesis (Fujita et al. 2006); however, further research is needed to draw these conclusions. Previous studies have shown that nutrient-restricted offspring often exhibit reduced plasma glucose levels due to impaired pancreatic development and limited hepatic glycogen stores (Green et al. 2010; Simmons et al. 2007). However, the current study indicated an increase in glucose concentration at PND60 in those lambs whose mothers have received citrulline supplementation. Elevation of glucose levels at this time point may be indicative of a more robust nutrient uptake from the small intestine and/or increased anabolic capacity to extract and utilize available nutrients. Alternatively, the increased glucose concentration at PND60 may indicate the development of insulin resistance, although other hallmarks of this type of metabolic imbalance, such as increased adiposity, were not observed in preweaning lambs. Nonetheless, further research is needed to elucidate the mechanisms by which circulating glucose levels are elevated in response to prior maternal citrulline supplementation and what the consequences of this might be at later stages of life.

Morphometric analyses of the pancreas in the present study revealed no difference in percentage of the endocrine or exocrine areas of the pancreas. Furthermore, the mass of both areas was increased in lambs from L-citrulline treated ewes. Acinar cells within the exocrine pancreas produce, store, and secrete digestive enzymes that are responsible for the hydrolysis of carbohydrates, fats, and protein (Bastida-Ponce et al. 2017; Leung et al. 2010). This increase in exocrine pancreas mass could demonstrate an increase in feed utilization, due to the secretion of enzymes from the exocrine pancreas utilized by the small intestine during the digestive process. The observation of an increase in small intestine mass in lambs from L-citrulline supplemented ewes could also indicate efficient utilization of feed, due to more surface area for nutrient absorption, potentially increasing muscle development and growth. Further research is needed to elucidate the correlation between exocrine enzyme secretion and metabolism during postnatal growth. In contrast, maternal citrulline supplementation tended to reduce pancreatic vascularization in the present study. This observation is interesting given that prior studies have observed stimulation of vascular function in the ovine placenta via arginine and citrulline supplementation (Gilbreath et al. 2020; Lassala et al. 2009; Wu et al. 2006). Further, in pigs, maternal citrulline or arginine supplementation was shown to promote vascularization of the placental membranes (Li et al. 2010, 2023). It has previously been reported that birth weight is positively correlated with the percentage of islet tissue (Fowden and Hill 2001). While there was no detectable increase in the endocrine pancreas percentage due to maternal L-citrulline supplementation, a tendency of increased δ-cell percentage was demonstrated in lambs from L-citrulline treated ewes. This could potentially increase glucose concentrations due to the inhibitory actions of somatostatin on β-cells (Bloom and Polak 1987; Huising et al. 2018). Previous IUGR models have demonstrated a reduction in β-cell mass (Green et al. 2010; Limesand et al. 2005, 2013; Simmons et al., 2007). The present study showed an increase in both β and α-cell mass in lambs from L-citrulline supplemented ewes, suggesting that maternal L-citrulline supplementation may enhance endocrine cell populations and support the development of metabolic regulatory capacity. In this regard, we found that the portion of endocrine cells positive for PCNA was increased in lambs from L-citrulline treated ewes. Limesand et al. (2005) observed no difference in β-cells positive for PCNA between IUGR and control fetus; however, an increase of PCNA positive α-cells was noted. While the present study did not analyze the proliferation of individual pancreas endocrine cell types, this increase suggests that L-citrulline plays a role in altering the length of endocrine cell cycles. With no difference in exocrine cell proliferation between groups, these results suggest that the expansion in pancreatic mass was likely driven by mechanisms such as increased proliferation rates at an early stage of pancreatic development.

To our knowledge, this is the first report that oral L-citrulline supplementation from gestational days 28 to 140 to nutrient-restricted ewes increases birth and weaning weights, while altering endocrine pancreatic function. Although the present study does not elucidate the mechanisms responsible for enhancing the development of key metabolic organs, our work provides potentially groundbreaking findings for future investigations.

In summary, results of the present study indicate that maternal dietary L-citrulline supplementation to nutrient-restricted ewes from days 28–140 of gestation may reduce the incidence of IUGR, alter function of the endocrine pancreas, and improve postnatal growth to weaning. Translation of these results into nutritional management strategies for gestating ruminants has significant potential to reduce incidences of morbidity and mortality and increase revenue at the time of product marketing. Future studies are warranted to determine at what stage during fetal development L-citrulline acts to promote pancreatic growth and target genes that are being affected during pancreas organogenesis. Finally, long-term follow-up beyond weaning would help determine whether the observed improvement in growth and pancreatic development translate into durable metabolic health advantages and enhanced efficiency of lean tissue gain. Thus, these findings may have important implications for improving growth, development, and health in humans and other mammals.

The present study investigated the effects of maternal L-citrulline supplementation during gestation on fetal growth, organ development, and early postnatal outcome in lambs born to nutrient restricted ewes. Key findings from the present study demonstrate that L-citrulline supplementation acts as a targeted nutritional intervention to support fetal development and metabolic function in offspring from IUGR pregnancies. Future research should focus on the effects of maternal L-citrulline supplementation on glucose tolerance and insulin sensitivity of the lambs in later life stages. Additionally, further research is warranted to elucidate the effects of maternal L-citrulline supplementation on feed efficiency and carcass cutability in lambs at time of slaughter. In conclusion, maternal dietary L-citrulline supplementation in nutrient-restricted pregnancies represents a promising strategy to improve fetal growth and postnatal pancreatic function. These findings contribute to the broader field of developmental programming and offer a foundation for translational research to reduce the long-term impacts of IUGR in both livestock and humans.

Competing Interests

The authors declare that they have no conflict of interest.

Funding

This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-37272 from the US Department of Agriculture’s National Institute of Food and Agriculture.

Author Contribution

Alissa Herring wrote the main manuscript text and prepared all figures and tables. Kyle Herron, London Lemcke, and M. Carey Satterfield aided in animal work and tissue collection. All authours reviewed the manuscript.

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No competing interests reported.

Maternal Dietary Citrulline Supplementation Increases Fetal Growth and Programs Pancreatic Development in the Lambs

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Animal Study and Tissue Collection

Determination of Circulating Insulin, Glucose, and NEFA Concentration

Determination of Pancreatic Histoarchitecture

Pancreatic Cell Proliferation

Morphometric Analyses

Statistical Analyses

Results

Discussion

Conclusion

Declarations

Competing Interests

Funding

Author Contribution

References

Additional Declarations

Status:

Version 1