Impact of Dietary Supplementation of L-Citrulline to Meat Goats During Gestation on Reproductive Performance

doi:10.21203/rs.3.rs-4426546/v1

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Impact of Dietary Supplementation of L-Citrulline to Meat Goats During Gestation on Reproductive Performance

https://doi.org/10.21203/rs.3.rs-4426546/v1

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Background

Meat goat production is a worldwide industry with products such as meat, milk, soap, and fiber being produced. There are approximately 2.6 million meat goats in the United States. For breeding female ruminants, early pregnancy loss is estimated to be 30% within the first month of gestation. Extracellular L-citrulline (a precursor to L-arginine) is not degraded by ruminal microbes due to the lack of uptake. L-arginine and thus L-citrulline, have beneficial impacts on placentation and, subsequently, fetal-placental development and survival.

Results

Meat goats were fed either a control (CON) or L-citrulline (CIT) supplemented diet from Days 12 to 82 of gestation. Blood samples were collected and subjected to high performance liquid chromatography analyses to quantify the abundance of amino acids. Pregnancy rates were determined on Days 30, 61, and 90 of gestation, and litter weight, individual birth weights, and 90-day adjusted weaning weights were collected. The concentrations of citrulline, ornithine, and arginine were greater in CIT does compared to CON does, but there was no difference in pregnancy rates between CON and CIT does. Birth weight was greater for male kids born as singles when compared to females, but this phenotype was not observed for kids born as twins or triplets. Further, males born to CON does had greater 90-day adjusted weaning weights than females, but this was not observed in the CIT group. Female kids born to CON and CIT Boer goats had heavier 90-day adjusted weaning weights than those born to Spanish or F1 Boer-Spanish does.

Conclusion

L-citrulline increased and stabilized circulating concentrations of arginine in maternal serum of meat goats. Further, L-citrulline supplementation may benefit does gestating triplets and increase 90-day adjusted weaning weights of female offspring. Boer goats may also be more sensitive to dietary supplementation with L-citrulline when compared to Spanish goats.

early pregnancy

meat goats

citrulline

arginine

amino acids

Meat goats are a popular source of protein in locations such as Asia, Africa, Mexico, Brazil, and the United States [1]. In the United States, there are 2.6 million meat goats with 38% of those animals residing in Texas [2]. Nutritionally, goat meat contains less fat and cholesterol than other animal meats and is an important source of protein and specific amino acids [1]. To ensure continued availability of goat meat for consumption around the world, it is vital that meat goat production continues to increase in efficiency. Unfortunately, early pregnancy loss in ruminant species is a concern, with a large percentage of these losses within the first month of gestation. Thus, there is potential to increase meat goat production if strategies are implemented to mitigate pregnancy loss [3].

L-citrulline is an amino acid and direct precursor to arginine [4]. Previous studies found that ruminal microbes do not degrade extracellular L-citrulline due to the lack of uptake, meaning that L-citrulline can be fed as an unprotected amino acid and absorbed in the small intestine where it is converted rapidly to arginine [5, 6]. Arginine has roles in cell signaling and reproductive processes including embryonic development, implantation, and placentation [7]. Arginine is also required for the production of nitric oxide (NO) and polyamines that directly impact angiogenesis, vasodilation, and the proliferation and migration of cells [4, 7]. Lassala et al. (2011) determined that intravenous administration of arginine to ewes from gestational Day 100 to 121 enhanced lamb survival and increased birthweights of quadruplets [8]. Further, another study observed that intravenous infusion of arginine from Day 60 of gestation until parturition prevented fetal growth restriction in undernourished ewes [9]. It has also been determined that the half-life of citrulline is longer than that for arginine, suggesting that L-citrulline is potentially a more impactful dietary supplement than arginine because it stays in the circulation longer [10].

The lack of degradation of dietary L-citrulline by ruminal microbes is a potential economic advantage to livestock producers, mainly because less processing of feedstuffs is required to protect this amino acid from ruminal microbial degradation. There is also ease associated with feeding L-citrulline because it can be mixed directly into rations and fed to the livestock. Therefore, the longer half-life and lack of degradation of extracellular L-citrulline in the rumen, in addition to the economic advantage of feeding this amino acid, makes it a potentially favorable supplement to enhance fetal growth and survival in meat goats and other ruminants. We hypothesized that feeding L-citrulline to gestating meat goats would increase concentrations of select amino acids (namely citrulline and arginine) in the maternal circulation to promote fetal development and enhance offspring performance. This hypothesis is based on evidence that dietary supplementation of L-arginine to pregnant pigs between Days 30 and 114 of gestation increased the number of live-born piglets by 2 per litter and litter birth-weight by 24% [11]. There is also evidence that dietary supplementation of pregnant rats with arginine increases the number of implantation sites and litter size by approximately three [12, 13]. Additionally, arginine supplementation improves fetal-placental growth and prevents intra-uterine growth restriction of fetuses in pregnant women [14].

Animals

Meat goats (Boer, Spanish, or F1 Boer-Spanish) were group housed at the International Goat Research Center at Prairie View A&M University, (Prairie View, Texas, USA; 30.100867, -95.977232) in a covered barn with grazing access and ad libitum access to clean water and feed. All experimental procedures followed the Guide for the Use of Agriculture Animals in Research and Teaching and were approved by the Institutional Animal Care and Use Committee at Prairie View A&M University.

Experimental animals and sample collection

Estrous cycles of reproductively mature meat goats (n = 97 were synchronized by inserting a CIDR (Zoetis, Parsippany, NJ, USA) and administering an injection of GnRH (1 mL Factrel, Zoetis). The does received an injection of PGF2α (1 mL Estrumate, Merck, Rahway, NJ, USA) 10 days later. CIDRs were removed 2 days after the PGF2α injection and the does were observed for estrus behavior. The following day, the does were injected with GnRH (1 mL Factrel, Zoetis) and bred via natural service 20 hours later.

Does were assigned to a breeding group (Groups 1–9) and then separated into treatment groups and fed either a L-citrulline supplemented diet [n = 45; 97.63% basal diet + 2% supplement (0.5% L-citrulline, 0.5% L-glutamine, 1% soybean hydrogenated oil) + 0.37% cornstarch] or an isonitrogenous control diet [n = 52; 97.63% basal diet + 1.37% L-alanine + 1% soybean hydrogenated oil] from Days 12 to 82 of gestation. Whole blood samples were collected from does via jugular venipuncture into vacutainer blood tubes without anticoagulant beginning on Day 12 of gestation and for every 14 days until Day 82 of gestation. A serial blood draw was performed with one group (n = 6 CON, n = 6 CIT) of does on Day 40 of gestation where whole blood samples were collected immediately before feeding (time 0) and at 30 min, and 1, 2, 4, and 6 hours after feeding. Collected samples were stored overnight at 4°C to clot and centrifuged the following day at 2,600 x g for 18 min at 5°C (Eppendorf 5920R, Hamburg, Germany). Serum was harvested and the samples stored at -20°C until analyzed using high performance liquid chromatography (HPLC).

Ultrasonography was performed on Days 30, 61, and 90 of gestation to confirm pregnancy. Following kidding, numbers of offspring (including the number of males and females) and birth weights were recorded. Kids were weaned at various ages ranging from 52 to 101 days of age. Therefore, a 90-day adjusted weaning weight was calculated.

Quantification of amino acids in serum

The abundances of 22 amino acids in serum were quantified using HPLC as described previously [15]. Only serum samples collected during the serial blood draw on Day 40 of gestation were analyzed using HPLC to determine systemic changes in concentrations of amino acids in maternal blood relative to time of feeding. Samples (100 µL) were deproteinized using 1.5 M HCLO₄ (100 µL) and 2 M K₂CO₃ (50 µL). The mixture was centrifuged at 10,000 x g for 3 min. The supernatant was collected and mixed with o-phthaldialdehyde (OPA) reagent to perform HPLC analyses using the method involving precolumn derivatization. The OPA reagent was generated by dissolving 50 mg OPA in 1.25 mL HPLC grade methanol (Fisher Scientific, St. Louis, MO, USA). Additionally, 11.2 mL of sodium borate (pH 9.5), 50 µL of 2-mercaptoethnol (M3148 Sigma Aldrich, St. Louis, MO, USA), and 0.5 mL of Brij-23 (B4184 Sigma-Alrich) was added to the mixture. The prepared sample mixture was composed of 1.4 mL of HPLC-grade water (Fisher Scientific), 100 µL of 1.2% benzoic acid (in 40 mM sodium borate, pH 9.5), and 100 µL of sample. In an autosampler (model 712 WISP, Waters, Milford, MA, USA), the assay mixture was derivatized with 30 mmol/L of the OPA reagent, and 15 µL of the mixture was injected into a Supelco 3 um reversed phase C18 column (150 x 4.6 mm inner diameter, Sigma-Alrich, St. Louis, MO). A solvent gradient was generated from solution A (0.1 M sodium acetate, 18% methanol, and 1% tetrahydrofuran, pH 7.2) and solution B (HPLC grade methanol) and used to separate amino acids. Amino acids were quantified relative to standards using Millenium 32 Software (Waters).

Statistical analyses

Pregnancy rates were calculated on Days 30, 61, and 90 by the following formula: (number of pregnant does/total number of does in the study)*100. Pre-weaning average daily gain (ADG) was calculated by subtracting birth weight from actual weaning weight and dividing by weaning age ([actual weaning weight – birth weight]/weaning age in days). Adjusted 90-Day Weaning Weight was calculated as follows: (Pre-weaning ADG*90) + birth weight). A contingency analysis was used to determine significance. Pearson’s correlation coefficient was the test statistic and chi-square ≤ 0.05 indicated significance.

A two-sample T test was applied to compare baseline concentrations of amino acids in maternal serum between treatment groups. Linear mixed effects model was used to fit the concentrations of amino acids in maternal serum over time and likelihood ratio tests were employed to assess the effects of treatments, group, litter size, breed of doe, fetal sex, and their interactions on various features including overall litter weight, birth weight, and 90-day adjusted weaning weights. Statistical significance was set at a P-value of ≤ 0.05. A P-value ≤ 0.10 indicated a trend towards significance. All data analysis was performed using JMP Pro (Version 16.0) and R Statistical Software (Version 4.3.2).

Concentrations of amino acids in serum

Concentrations of citrulline, arginine, ornithine, and alanine in maternal serum at different time points after consuming a meal for the CON and CIT groups are summarized in Table 1. Concentrations of 18 other amino acids are summarized in Table 2. The concentrations of citrulline (P < 0.001) and ornithine (P = 0.05) decreased over time in both the CON and CIT treated does, but the CIT does had greater concentrations of these amino acids in serum than CON does (P < 0.05; Table 1). Concentrations of arginine in maternal serum decreased for CON does but were stable in CIT-fed does. Further, there was a tendency for concentrations of arginine to be greater for CIT-fed as compared to CON-fed does (P < 0.1; Table 1). The concentrations of alanine were lower in CON does compared to CIT does (P < 0.05; Table 1) but remained stable over time.

The concentrations of tyrosine, tryptophan, valine, phenylalanine, isoleucine, leucine, lysine and asparagine were typically greater in the CIT-fed does as compared to CON-fed does, except for valine and phenylalanine (P < 0.05; Table 2). There was also a tendency for the concentrations of histidine and threonine to be greater in CIT-fed than CON-fed does (P < 0.1; Table 2). The concentrations of glycine and taurine decreased over time, while those for beta-alanine increased over time (P < 0.05; Table 2). There was also a tendency for threonine to decrease over time in both the CON and CIT does (P < 0.1; Table 2).

Litter weight, birth weight, and adjusted 90-day weaning weight

The overall pregnancy rates at Days 30, 61, and 90 of gestation were 88.5%, 86.5%, and 85.6%, respectively. On Day 90 of gestation, the pregnancy rate for CON does was 90.6% and 80.4% for CIT does. Pregnancy rates between CON and CIT-fed does were not statistically different on Days 30 or 90 of gestation (P > 0.05); however, on Day 61 of pregnancy, there tended to be more non-pregnant CIT does than CON does (Prob > ChiSq = 0.072).

Litter weights, obtained by calculating the sum of birth weights after the kids were born, were not affected by CON and CIT diets. Interestingly, there were four does in the CON group that had quadruplets, but none in the CIT group. Dietary L-citrulline supplementation did not affect litter size or birth weights of the kids (Fig. 1A). Single kids born to CON does were heavier than single kids born to CIT does (P < 0.05), but there was no treatment effect on birth weights for kids born as twins or triplets (Fig. 1B). Further, when accounting for sex of the offspring and number of kids born per doe, birth weights of single and twin males born to CON does tended to be greater than those born to CIT does (P < 0.1), but this phenotype was not observed in males born into a litter of triplets (Fig. 1C). Additionally, birth weights for female offspring were not affected by litter size.

There was no difference in 90-day adjusted weaning weights between kids born to CON does and CIT does; however, breed of doe affected 90-day adjusted weaning weights (P < 0.05, Fig. 2A and C). When not accounting for breed of doe, but considering effect of sex of the kids, 90-day adjusted weaning weights were greater for male kids born to CON does as compared to female kids born to CON does, but this phenotype was not observed for kids born to CIT does (P < 0.05; Fig. 2B). Finally, when accounting for the effect of breed, 90-day adjusted weaning weight was greater for CIT males born to Boer does, and there was a tendency for it to be greater for female kids born to CIT does when compared to the CON kids (P = 0.0502, Fig. 2C); however, this phenotype was not observed for Spanish or Boer-Spanish F1 does.

Citrulline is a direct precursor for arginine and, thus, likely contributes to the production of NO and polyamines which have beneficial effects on conceptus growth and survival [4, 16]. Extracellular citrulline is not degraded by ruminal microbes, meaning that it can be fed as an unprotected amino acid as a proxy for arginine with nutritional benefits [5, 6]. This study aimed to determine the systemic concentrations of select amino acids in does fed L-citrulline from Day 12 to 82 of gestation and the subsequent performance of their offspring.

Results of the present study indicated that concentrations of citrulline and ornithine (a product of arginine hydrolysis by arginase) were greater in the CIT-fed group (P < 0.05) but decreased overtime in both the CON and CIT groups (P < 0.05). Interestingly, concentrations of arginine in maternal plasma decreased over time in CON does, but not in CIT does. The concentrations of arginine tended to be greater in CIT-fed does (P = 0.092), suggesting that L-citrulline can stabilize systemic concentrations of arginine in pregnant meat goats. These findings support those from other studies indicating that concentrations of arginine are greater in the plasma of sheep and cattle after consumption of L-citrulline [5, 6]. Further, sheep that consumed rumen protected arginine had greater concentrations of arginine in plasma [17].

The concentrations of alanine, tyrosine, tryptophan, isoleucine, leucine, lysine, and asparagine were lower for CON does than CIT does (P < 0.05). More studies are needed to determine the potential roles of these amino acids in gestating meat goats. However, those amino acids are not likely involved in the same metabolic pathways as citrulline and arginine but may be degraded via the pathways that are regulated by arginine and its metabolites.

There was no significant difference in pregnancy rates between CON and CIT groups (P > 0.05). Thus, feeding L-citrulline neither enhances nor hinders pregnancy outcomes in meat goats and citrulline fed at the current dose had no detrimental effects on gestating meat goats. This finding indicates that L-citrulline is a safe supplement and has the potential to be used in international meat goat enterprises. In previous studies, it was suggested that the presence of 1% soybean hydrogenated oil (originally used to encapsulate citrulline) in the diet, may interfere with the action of citrulline, arginine, or its metabolites (such as NO, polyamines, and creatine) in some ruminant species (e.g., sheep) through unknown mechanisms [5, 6, 17].

Overall, birth weights were unaffected by treatment; however, single kids born to CIT-fed does were lighter than single kids born to CON-fed does (P < 0.05). There was no difference in birth weights among treatment groups for twins and triplets, suggesting that L-citrulline supplementation could be of greater benefit for does gestating multiple fetuses. This observation is consistent with findings of Lassala et al. (2011), that intravenous administration of arginine enhanced growth of lambs in ewes with twin or quadruplet pregnancies [8].

The 90-day adjusted weaning weights were greater for males born to CON does when compared to females born to CIT does (P < 0.05), but this effect was mitigated in does fed L-citrulline. Further, when the effects of diet were evaluated based on breed of doe, it was observed that for Boer goats, males born to CIT does were heavier than males born to CON does. Similarly, females born to Boer goats in the CIT group tended to be heavier than those in the CON group (P = 0.0502). The same results were not observed for Spanish or Boer-Spanish F1 does, suggesting that Boer goats are potentially more sensitive to the L-citrulline dietary supplementation than the other breeds of meat goats in this study. This is an interesting finding as the protein and energy requirements for growing Boer and Spanish influenced goats are not known to be different [18, 19]. It has also been suggested that milk from Boer goats is higher in energy content than milk from other breeds of goats; however, further studies are needed to determine if there is a correlation between L-citrulline supplementation, milk yield, and weaning weights of meat goat kids [20].

Results from this study indicate that feeding unprotected L-citrulline to meat goats from Days 12 to 82 of gestation can increase and stabilize circulating levels of arginine in pregnant does. Further, these results suggest that dietary L-citrulline supplementation may be of benefit to does gestating multiple fetuses and that it can increase 90-day adjusted weaning weights for female offspring compared to males. Interestingly, Boer goats appeared to be more sensitive to this supplementation. Collectively, this study revealed that the inclusion of L-citrulline in diets of pregnant meat goats may be beneficial for improving fetal growth and overall reproductive performance. For livestock producers, the benefit of feeding this supplement is due to the fact that extracellular L-citrulline escapes ruminal degradation; therefore, processing it and mixing it into rations is less expensive than for rumen protected L-arginine. Additionally, the longer half-life of L-citrulline compared to L-arginine is advantageous to both livestock and humans. A major limitation of this study was the need to group feed does on the respective treatments. Therefore, the exact intake of dietary L-citrulline could not be determined. Future studies of meat goats fed individually are needed for dose-response studies to better define optimal rates of the dietary supplementation of L-citrulline. Additionally, the underlying mechanisms involved in and impacted by L-citrulline supplementation and its impact on the reproductive performance of meat goats, other livestock animals, and humans should be investigated.

CON – control-fed group

CIT – citrulline-fed group

NO – nitric oxide

HPLC – high performance liquid chromatography

OPA – o-phthaldialdehyde

ADG – average daily gain

Ethics approval and consent to participate

All experimental procedures followed the Guide for the Use of Agriculture Animals in Research and Teaching and were approved by the Institutional Animal Care and Use Committee at Prairieview A&M University.

Consent for publication

Not applicable.

Availability of data and material

The datasets presented in this paper can be made available by the corresponding author upon request.

Competing interests

The authors have no conflict of interest to declare.

Funding

This research was supported by Texas A&M Agrilife Research and Prairie View A&M Cooperative Agricultural Research.

Authors’ contributions

The experiments were planned by FWB, WBF, and GW and executed by MGN with assistance from ANL, CS, KLH, EDC, XL, and LZ. The first draft of the manuscript was written by MGN and FWB and edited by ANL, CS, KLH, EDC, XL, LZ, GW, and WBF. The final version of the manuscript was prepared by MGN and FWB and the authors read and approved the final manuscript.

Acknowledgements

The authors would like to acknowledge the research and animal care staff at Prairie View A&M University in addition to the undergraduate students, graduate students, faculty, and staff at both universities that made these experiments possible.

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Table 1 and 2 are available in the Supplementary Files section.

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Editorial decision: Major revision
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You are reading this latest preprint version

Impact of Dietary Supplementation of L-Citrulline to Meat Goats During Gestation on Reproductive Performance

Status:

Version 1

Abstract

Background

Results

Conclusion

Figures

Background

Methods

Animals

Experimental animals and sample collection

Quantification of amino acids in serum

Statistical analyses

Results

Concentrations of amino acids in serum

Litter weight, birth weight, and adjusted 90-day weaning weight

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Version 1