Dietary Citrulline Supplementation Enhances Milk Production in Lactating Dairy Goats

doi:10.21203/rs.3.rs-4426614/v1

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Dietary Citrulline Supplementation Enhances Milk Production in Lactating Dairy Goats

https://doi.org/10.21203/rs.3.rs-4426614/v1

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Background Nutrition, day of lactation, litter size, parity, and sire impact lactational performance in goats. Arginine (Arg) has important roles in synthesis of nitric oxide (NO), polyamines, and creatine. Ruminal microbes degrade extracellular Arg; however, extracellular Cit is not degraded by ruminal microbes and can be fed unencapsulated as a proxy for Arg. Cit is absorbed in the small intestine, converted to Arg, then metabolized to NO, polyamines and creatine that may enhance lactational performance. This study determined effects of dietary citrulline (Cit) supplementation on milk production and milk composition of Alpine dairy goats. Does were synchronized to estrus and bred to Alpine bucks. Parturition was induced on Day 149 of gestation. After kidding, does were suckled overnight to allow their kid(s) to obtain colostrum before being milked 24h later (Day 1 of lactation). Does were assigned to either control (CON, n = 24) or citrulline (CIT, n = 23) supplemented diets. The isonitrogenous control diet was supplemented with 1.37% alanine and 1.00% soybean hydrogenated oil. The CIT supplemented diet was 97.63% basal diet with a 2.37% supplement (0.5% Cit, 0.5% Glutamine, 1% soybean hydrogenated oil, and 0.37% cornstarch). Diets were group fed ad-libitum by treatment group. Blood samples were collected on Days 0 and 30 of lactation, and daily milk volumes were collected twice daily. On Days 10, 20, and 40 of lactation, milk samples were collected for compositional analyses.

Results CIT-treated does had greater mean daily milk production (P = 0.0332) and there was an effect of day of lactation on mean daily milk production (P < 0.0001). Does producing three kids had greater mean daily milk production than does producing one kid (P<0.001). Multiparous does had greater mean daily milk production than primiparous does (P<0.0001), and there was an effect of sire on mean daily milk production (P<0.05). Compositional analyses revealed that Cit supplementation increased soluble-non-fat (SNF) (P= 0.0189) and protein (P=0.0238) in milk.

Conclusions Dietary supplementation of Cit fed ad-libitum increased mean daily milk yield and impacted milk composition in Alpine does. Further investigations should seek to understand underlying mechanisms responsible for these effects.

Lactation

Milk Composition

Arginine

Citrulline

Dairy Goats

Lactational performance of dairy goats depends on multiple factors such as nutrition, breed, overall health, age, and environment. For Alpine dairy goats, the period of lactation can be more than 240 days [1]. Daily milk production and milk composition during the course of the lactation curve change depending on breed, stage of lactation, environment, nutrition, health, and other factors affecting dairy goats. Milk composition for Alpine dairy does averages approximately 2.46% fat, 2.79% protein, 4.17% lactose, and 7.5% solids not fat (SNF) [2]. The milk goat industry is a vital agricultural enterprise in many countries, including the United States, the Mediterranean region, and China [3]. The composition of goat milk differs from that of milk from cows and women in having greater digestibility [4]. Of note, the consumption of goat milk is beneficial to individuals with allergens and gastro-intestinal disorders due to its lower content of lactose in comparison to cow’s milk [5]. As the dairy goat industry continues to grow globally, it is important to utilize management techniques to improve productivity. As new knowledge from research is available, it can be translated into management strategies in the dairy industry to improve productivity and profitability.

Nutritional status of females is a factor that greatly impacts milk production and composition, so it is imperative to optimize diets in order to optimize quantity and quality of milk produced. In ruminants, hydrolysis and fermentation of nutrients occurs in the rumen by enzymes, bacteria, and microbes [6]. Ruminants such as goats, cows, and sheep have a rumen, reticulum, omasum, and abomasum in comparison to the simple stomach in monogastric species [7]. The rumen is lined by papillae and inhabited by bacteria (95% of the microbial population), archea, fungi, and protozoa [8]. These microbes have multiple roles in digestion of proteins, degradation of amino acids (AA) degradation, and protein synthesis by microbes [8, 9]. Protein metabolism is the direct result of metabolic activity of ruminal microbes [10]. It has been suggested that AA uptake by ruminal microbes may be the limiting factor in protein degradation in the rumen [10]. Once AAs are taken up by ruminal microbes, they are incorporated into protein or are broken down into ammonia for synthesis of AA [9–12]. In ruminant nutrition, a long-standing view is that all dietary AAs, including Arg and glutamine, undergo extensive degradation by ruminal microbes [13, 14]. However, it has been discovered that citrulline (Cit) is not degraded by the ruminal microbes of ruminants including sheep and cattle [15–17]. Therefore, it may be directly supplemented in the diet without the need for encapsulation or protection to serve as a proxy for Arg. Endogenous Arg in ruminants is synthesized by ruminal bacteria and from citrulline (Cit; formed from glutamine/glutamate and proline in enterocytes of the small intestine) by extrahepatic tissues [18]. Arg is a common substrate for synthesis of nitric oxide (NO), polyamines and creatine [14]. Arg also has important roles in other metabolic pathways including ammonia detoxification and creatine metabolism. Arg metabolism has important roles to improve reproductive performance in swine [19]. Arg metabolism is highly compartmentalized and requires inter-organ cooperation as not all of the enzymes are expressed in all cells and tissues [14]. The major site of Arg synthesis from Cit is the kidneys, and the intestine has a unique capacity for Cit synthesis from glutamine and proline [20]. Cit has important roles for regulation of nitrogen homeostasis, immune responses, and the cardiovascular system [21]. The intestinal-renal axis allows synthesis of Cit from glutamate and glutamine in the small intestine and its conversion to Arg in the kidneys [22]. Glutamine is first converted into glutamate by glutaminase, which is then catabolized into Cit [23]. Subsequently, enterocytes release Cit into the portal circulation for transport to the kidneys where it is converted into arginosuccinate [23]. Arginosuccinate is then converted into fumarate and then Arg via argininosuccinate lyase [23]. Therefore, increases in concentrations of Cit may increase concentrations of Arg, as Cit is a proxy for the production of Arg. Therefore, in ruminants, Cit may supplemented unencapsulated for absorption by the small intestine and use for Arg synthesis, thus increasing the availability of endogenous Arg for synthesis of NO, polyamines and creatine [15, 24]. The utilization of endogenous Arg, specifically in support of lactation, emphasizes its multifunctional properties. Based on the foregoing, we hypothesized that dietary Cit supplementation would increase the availability of Arg to improve milk production and milk composition in lactating dairy goats. Gaining insight into the impacts of dietary supplementation of Cit on lactational performance and milk composition was expected to suggest new management strategies to improve milk production and composition to benefit both dairy goat and dairy cattle industries.

Animals

Lactating alpine dairy goats were utilized to study the effects of dietary supplementation of L-citrulline (N = 47). All goats were fed to meet National Research Council requirements and assigned randomly to either the control treatment group (CON, n = 24), or the L-citrulline treatment group (CIT, n = 23) [25]. The does assigned randomly to the CON group received a supplementation of 1.37% alanine, as an isonitrogenous control, and 1% soybean hydrogenated oil. Does assigned to CIT group received a 2.37% supplement (0.5% Cit, 0.5% glutamine, 1% soybean hydrogenated oil, and 0.37% cornstarch). All does in each treatment group were housed together and group fed with free access to feed and water throughout the study.

Experimental Design and Sample Collection

On Day 146 of gestation a single dose of 0.5 ml Estrumate (cloprostenol; Merck, Rahway NJ) was administered followed by a second dose of 0.25 ml Estrumate (cloprostenol; Merck, Rahway NJ) the following morning on Day 147 to induce parturition on approximately Day 149 of gestation. Following parturition (designated Day 0 of lactation), does were allowed to nurse their kid(s) overnight to obtain colostrum. Each doe entered the milk line 24 h following kidding, which was designated Day 1 of lactation. The randomly assigned treatment supplementation began on Day 1 of lactation and continued through Day 40 of lactation. All alpine does were milked twice daily with mechanical milkers and daily production of milk (liters) recorded by DeLaval milk meters for each doe at each milking. Volumes (liters) of milk produced on Days 0, 10, 20, and 40 were utilized to determine effects of Cit dietary supplementation on milk production. One ounce of milk was collected on Days 10, 20, and 40 of lactation for compositional analyses. On Days 0 and 30 of lactation, blood samples were collected in 10 mL BD vacutainer blood collection tubes from each doe via jugular collection, stored at 4°C overnight. Blood samples were centrifuged (Eppendorf centrifuge 5920R Hamburg, Germany) at 5°C for 18 min at 2,600 x g. Serum was harvested and stored at -20°C until analyzed for concentrations of AA using high pressure liquid chromatography (HPLC) analyses [26].

Compositional Analyses

Milk samples collected on Days 10, 20, and 40 of lactation were allowed to cool to room temperature after collection. Once at room temperature, a somatic cell count was performed using a DeLaval DM SCC counter (DeLaval lnc., Tumba, Sweden). The raw milk samples were then analyzed using a Page and Pederson Lactometer (LactiCheck-02 Rapi Read Page & Pederson Int.) for determination of butter fat, protein, lactose, and solids not fat (SNF).

Analysis of Amino Acids

Effects of dietary supplementation of Cit on concentrations of amino acids in serum were determined using HPLC and modified procedures described previously [27]. Each serum sample (100 µL) was acidified with 100 µL of 1.5 mol/L HCLO₄ and vortexed. The acidified sample was then neutralized with 50 µL of 2 mol/L K₂CO₃ and vortexed. The neutralized sample was then centrifuged for 3 min at 10,000 x g in an Eppendorf centrifuge (Eppendorf centrifuge 5920R). The neutralized supernatant was then collected and diluted 10-fold before analyzed by HPLC using precolumn derivatization o-phthaldialdehyde (OPA) reagent II. The OPA reagent II was prepared by dissolving 50 mg OPA in 1.25 mL HPLC-grade methanol, followed by 11.2 mL of sodium borate (pH 9.5), 50 µL of 2-mercaptoethanol, and 0.5 mL of Brij-23 (Sigma Aldrich, St. Louis, MO). The assay mixture contained 1.4 mL of HPLC grade water (Fisher Scientific, Hampton, NH), 100 µL of 1.2% benzoic acid (in 40 mmol/L sodium borate, pH 9.5), and 100 µL of sample. The assay mixture was derivatized in an autosampler (model 712 WISP, Waters, Milford, MA) with 30 mmol/L OPA, and 15 µL of the derivatized mixture was injected into a Supelco 3-um-reverse-phased C18 column (150 mm X 4.6 mm inner diameter, Sigma-Aldrich, St. Lois, MO). Amino acids were separated by a solvent gradient comprised of solution A (0.1 mol/L sodium acetate, 18% methanol, and 1% tetrahydrofuran, pH 7.2) and solution B (100% methanol). Concentrations of amino acids in the serum samples were quantified relative to authentic standards using Millennium-32 Software (Waters, Milford, MA).

Statistical Analyses

Statistical analyses of data using the unpaired t-test or one-way ANOVA were performed using JMP (Version JMP Pro 16). The normality of the distribution of daily milk production throughout the lactation curve was assessed using a goodness-of-fit test, and a P value of ≤ 0.05 indicated that the data were not normally distributed. For data that were not normally distributed, a nonparametric Wilcoxon/Kruskal-Wallis test was used for mean comparisons. Pair-wise comparisons of means for data that were not normally distributed were made using the non-parametric Wilcoxon each-pair comparison test. The test of one-way ANOVA was completed for data with a normal distribution. The mean daily milk production data were normally distributed (P = 0.248). To compare means on a pair-wise comparison basis for data normally distributed, the each-pair student’s T test was used. Least squares regression analyses were completed to determine interactions among treatment, day of lactation, litter size, number of parities, and sire on daily mean milk production. Normality of mean daily milk production from each doe was assessed using the goodness-of-fit test. The effects of fetal sex on mean daily milk production for each doe were assessed using the nonparametric Wilcoxon/Kruskal-Wallis test to compare means. The comparison of multiparous and primiparous does on mean daily milk production was assessed using a one-way ANOVA test. Interactions among other factors were evaluated using least squares-regression analyses. The normality of concentrations of AA in serum samples collected on Days 0 and 30 of lactation were assessed using goodness of fit tests. The concentrations of AA were normalized to values from Day 0 of lactation for each doe which was a baseline and factor of 1 to which concentrations of AA in serum on Day 30 of lactation were compared (concentration of AA on Day 30/concentration of AA on Day 0). A univariate repeated measures test was used to analyze those data to determine the effect of treatment over the 30-day period of lactation. A univariate repeated measures test accounts for day of supplementation while capturing correlations of AA concentrations within each doe [28]. This method considers Day 0 of lactation baseline concentrations as a covariate and uses the difference from baseline as a method to normalize data [28]. In all methods of statistical analyses, results were considered statistically significant at P ≤ 0.05, trending towards significance at 0.05 < P < 0.10, and not significant at P ≥ 0.10.

Effects of Cit Supplementation, Litter Size, Parity, and Sire on Milk Production

A one-way ANOVA revealed that CIT does had greater milk production than CON does (P = 0.04) (Fig. 1), and that the litter size of does influenced milk production (P = 0.03) (Fig. 2). On a pair-wise comparison basis, does producing three kids produced more milk than does producing either 1 (P = 0.01) or 2 (P = 0.03) kids.

One-way ANOVA indicated that multiparous does produced more milk than primiparous does (P < 0.0001) (Fig. 3) and sire affected milk production by does (P = 0.002) (Fig. 4). No significant interactions between treatment and litter size, parity, and sire were detected. However, multiparous does in the CIT treatment group tended to have greater milk production than multiparous does in the CON group (P = 0.058), but there was no treatment effect among primiparous does.

Comparison of milk production across days of lactation

Mean comparisons of milk production across days were assessed using the Wilcoxon/Kruskal-Wallis test that revealed an effect of day of lactation on milk production (P < 0.0001). A non-parametric pairwise comparison among days indicated that milk production on Day 40 of lactation was greater than that on Day 0 (P < 0.0001) and Day 10 (P = 0.041) of lactation. Milk production on Day 20 of lactation was greater than that on Day 0 (P < 0.0001) of lactation, and milk yield on Day 10 of lactation was greater than that on Day 0 of lactation (P < 0.0001). These results are presented in Fig. 5.

Compositional analysis of milk samples

Mean concentrations of protein in milk collected on Days 10, 20, and 40 of lactation were greater in the milk from CIT than CON does (P = 0.024), and there was a trend (P = 0.083) for greater lactose content in milk from CIT than CON does. Treatment did not affect fat content in milk at any stage of lactation investigated (P > 0.10). Milk collected on Days 10, 20, and 40 of lactation from CIT does had greater mean concentrations of SNF than milk from CON does (P = 0.019) throughout the lactation curve. Data on mean composition of milk samples and their lactose, fat, protein, and SNF content are presented in Fig. 6.

Concentrations of amino acids in serum

There was no effect (P > 0.10) of dietary Cit supplementation on concentrations of Arg, Cit, Orn, and alanine in serum (Table 1). The mean concentrations of aspartate, glutamate, asparagine, serine, glutamine, histidine, glycine, threonine, β-alanine, taurine, tyrosine, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine, and lysine in serum of goats on Days 0 and 30 of lactation resulted in no significant effects (P > 0.10) of Cit supplementation and a summary of these concentrations are presented in Table 2.

Table 1

Concentrations of citrulline, arginine, ornithine, and alanine in serum of lactating goats on Days 0 and 30 of lactation. Mean values (nmol/mL) \(\:\pm\:\:SD\:\)at a 10X dilution factor. The numbers of does are 24 and 23 for the Control and Citrulline fed groups, respectively. **Day Control (alanine supplementation) Citrulline supplementation**
	Cit	Arg	Orn	Ala		Cit	Arg	Orn	Ala
0	87 \(\:\pm\:\:49\)	211 \(\:\pm\:\:42\)	37 \(\:\pm\:\:26\)	182 \(\:\pm\:\:46\)		92 \(\:\pm\:\:42\)	213 \(\:\pm\:\:29\)	31 \(\:\pm\:\:11\)	219 \(\:\pm\:\:65\)
30	184 \(\:\pm\:\:68\)	217 \(\:\pm\:\:60\)	74 \(\:\pm\:\:38\)	220 \(\:\pm\:\:58\)		150 \(\:\pm\:\:53\)	219 \(\:\pm\:\:29\)	65 \(\:\pm\:\:20\)	239 \(\:\pm\:\:73\)

Table 2

Concentrations (nmol/mL \(\:\pm\:\:SD)\) of aspartate, glutamate, asparagine, serine, glutamine, histidine, glycine, threonine, β-alanine, taurine, tyrosine, tryptophan, methionine, valine, phenylalanine, isoleucine, leucine, and lysine in serum of goats on Days 0 and 30 of lactation while consuming diets supplemented with alanine or citrulline at a 10X dilution factor. The numbers of does are 24 and 23 for the CON and Cit groups, respectively. **Day Control (alanine supplementation) Citrulline supplementation**
	Asp	Glu	Asn	Ser	Asp	Glu	Asn	Ser
0	1.8 \(\:\pm\:\:1.7\)	120 \(\:\pm\:\:36\)	41 \(\:\pm\:\:17\)	90 \(\:\pm\:\:36\)	2.7 \(\:\pm\:\:1.8\)	131 \(\:\pm\:\:39\)	38 \(\:\pm\:\:8\)	62 \(\:\pm\:\:24\)
30	2.6 \(\:\pm\:\:1.9\)	140 \(\:\pm\:\:35\)	51 \(\:\pm\:\:18\)	140 \(\:\pm\:\:39\)	3.8 \(\:\pm\:\:1.9\)	138 \(\:\pm\:\:42\)	53 \(\:\pm\:\:18\)	111 \(\:\pm\:\:31\)
	Gln	His	Gly	Thr	Gln	His	Gly	Thr
0	332 \(\:\pm\:\:77\)	72 \(\:\pm\:\:14\)	483\(\:\pm\:\:162\)	50 \(\:\pm\:\:21\)	329 \(\:\pm\:\:53\)	73 \(\:\pm\:\:13\)	537 \(\:\pm\:\:163\)	59 \(\:\pm\:\:23\)
30	349 \(\:\pm\:\:67\)	57 \(\:\pm\:\:19\)	\(\:66\pm\:167\)	47 \(\:\pm\:\:23\)	375 \(\:\pm\:\:104\)	65 \(\:\pm\:\:11\)	691\(\:\pm\:\:272\)	55 \(\:\pm\:\:29\)
	β-Ala	Tau	Tyr	Trp	β-Ala	Tau	Tyr	Trp
0	0.8 \(\:\pm\:\:0.2\)	163 \(\:\pm\:\:37\)	54 \(\:\pm\:\:25\)	34 \(\:\pm\:\:13\)	1.8 \(\:\pm\:\:0.8\)	199 \(\:\pm\:\:49\)	49 \(\:\pm\:\:8\)	33 \(\:\pm\:\:9\)
30	1.1 \(\:\pm\:\:0.6\)	149 \(\:\pm\:\:53\)	60 \(\:\pm\:\:25\)	35 \(\:\pm\:\:16\)	1.7 \(\:\pm\:\:0.9\)	134 \(\:\pm\:\:21\)	55 \(\:\pm\:\:19\)	38 \(\:\pm\:\:9\)
	Met	Val	Phe	Ile	Met	Val	Phe	Ile
0	34 \(\:\pm\:\:13\)	165 \(\:\pm\:\:88\)	47 \(\:\pm\:\:23\)	117 \(\:\pm\:\:58\)	32 \(\:\pm\:\:11\)	178 \(\:\pm\:\:36\)	50 \(\:\pm\:\:10\)	106 \(\:\pm\:\:40\)
30	35 \(\:\pm\:\:16\)	178 \(\:\pm\:\:35\)	50 \(\:\pm\:\:11\)	140 \(\:\pm\:\:71\)	38 \(\:\pm\:\:9\)	172 \(\:\pm\:\:54\)	43 \(\:\pm\:\:12\)	116 \(\:\pm\:\:36\)
	Leu	Lys			Leu	Lys
0	161 \(\:\pm\:\:64\)	138 \(\:\pm\:\:49\)			151 \(\:\pm\:\:62\)	122 \(\:\pm\:\:27\)
30	140 \(\:\pm\:\:65\)	156 \(\:\pm\:\:71\)			169 \(\:\pm\:\:43\)	145 \(\:\pm\:\:31\)

This study was designed to test the hypothesis that dietary supplementation of unencapsulated Cit would enhance lactational performance and milk composition as Cit would be converted to Arg which would then increase Arg availability to increase milk production. This hypothesis was formed based on results of previous studies demonstrating beneficial effects of Cit and Arg metabolism on reproductive performance and lactational performance in pigs [29, 30]. Similar to swine, the enterocytes of adult cattle have increased rates of intestinal synthesis of Cit and Arg from glutamate and Pro during pregnancy [31]. Also, dietary supplementation with Cit enhances endogenous synthesis of Arg in ruminants [15, 16]. In ruminants, Cit derived from the small intestine is locally converted into Arg via the intestinal-renal axis [31]. In fed sheep, the small intestine releases Cit into the vasculature and the kidneys uptake approximately 1.41mmol/h [31]. Subsequently, the kidneys release Arg at approximately 1.46 mmol/h [31].

In ruminants, such as cows, goats, and sheep, virtually all dietary unencapsulated Arg is rapidly degraded in the rumen and does not reach the small intestine for absorption [31]. However, extracellular Cit is not subject to microbial uptake and degradation in the rumen of cattle and can, therefore, be taken up in the small intestine [32]. Transporters for Cit, solute carrier family members SLC38A3 and SLC38A5, are not expressed by ruminal microbes so Cit bypasses microbial degradation, unlike Arg which is rapidly degraded by ruminal microbes [33]. Cit transport is tissue- and cell-specific, and includes AA transporters, common transport system with Arg, L, N, Β⁰,B^0,+, and b^0,+ transporters that depend on tissue and cell type [34]. Studies with sheep and cattle found no detectable uptake of ¹⁴C-labeled Cit by ruminal microbes indicating that Cit may be supplemented without encapsulation and will not be degraded by ruminal microbes [17]. Ewes fed a diet supplemented with Cit had greater concentrations of Cit, Orn, and Arg that increased linearly with increasing doses of Cit supplementation [35]. Concentrations of NO in serum were also 11.25% greater in the Cit supplemented ewes compared to ewes fed a control diet [35]. Therefore, available results from studies in ruminants indicate that dietary Cit may be supplemented unencapsulated as a proxy for Arg as it is absorbed by the small intestine, then converted into Arg and utilized for synthesis of NO, polyamines, and creatine as well as being utilized in other metabolic pathways. In gestating gilts, dietary Cit supplementation from Days 14 to 25 of gestation improved placental synthesis of NO and polyamines, as well as angiogenesis to improve embryonic development [30]. These results indicate that through dietary supplementation of Cit, endogenous synthesis of Arg was increased for enhancement of synthesis of NO and polyamines in swine [30].

In our current study, dietary supplementation of unencapsulated Cit increased daily milk production, which supports our hypothesis. Higher milk yield and protein concentrations in milk in the Cit-supplemented goats could indicate a greater availability of not only Arg but also other AAs for protein synthesis by the mammary glands of the lactating goats, as compared with the CON group. The lack of increases in concentrations of Cit and Arg in the serum of the Cit group may result from a greater transfer of AAs from maternal blood to the mammary glands. Future studies with isotopes are warranted to test this hypothesis. Nonetheless, our results suggest that the dietary Cit supplementation likely increased Arg availability for production of NO and polyamines. These bioactive molecules contribute to increases in blood flow and rate of transport of nutrients and molecules to the mammary gland and associated tissues, thus improving cellular functions and overall milk production. As no significant interactions between treatment and litter size, parity, day of lactation, or sire were found, it seems that the effects of dietary Cit supplementation were independent of those factors.

The effect of litter size on milk production is positively correlated as expected [36]. An increase in litter size would increase placental mass and, in turn, an increase in placental lactogen associated with alveolar development in the mammary glands during pregnancy [36]. That study also revealed a significant effect of litter size on mean daily milk production, which aligns with other results in the literature [36]. In addition, no significant interaction between treatment and litter size in the present study was detected. In contrast, there was an effect of parity on milk production by lactating goats, which is consistent with results from research with ewes indicating that those with 2 and 3 parities had greater milk yield than primiparous ewes [37]. Our present study utilized 13 alpine bucks and there was a significant effect of sire on milk production. This result also aligns with literature, which emphasizes the effects of sire of fetus on milk yield in cattle [38]. Collectively results of this study align with the literature and support our hypothesis as dietary supplementation of L-Cit increased daily mean milk production and enhanced the composition of milk from lactating alpine dairy goats.

The results of this study support our original hypothesis that dietary supplementation with Cit would enhance lactational performance and milk composition. Dietary supplementation of Cit also increased SNF and protein content of goat milk and tended to increase lactose content in the milk. In this study, effects of day of lactation, litter size, parity, and sire were all consistent with effects reported for other species with each having a significant effect on mean daily milk production. These results reveal beneficial management strategies which could improve production, efficiency, and overall profits in the dairy goat industry. This study was limited to the use of does that were group fed ad-libitum either the control diet or Cit-supplemented diet which is how this management strategy would be practiced on a commercial dairy goat enterprise and as a common practice in other livestock production programs with ruminants. However, future studies should be conducted to determine optimal dosage of dietary Cit supplementation within the diet to elicit optimal effects. investigation into concentrations of AA in serum should also be conducted through serial blood collections after feeding to detect effects of Cit dietary supplementation on circulating concentrations of AA. These results suggest that dietary supplementation of Cit fed ad-libitum may be utilized as a proxy for Arg to increase the synthesis of NO, polyamines and creatine to enhance lactational performance and elicit changes in milk composition. Further, mechanistic studies on inter-organ cooperation responsible for the effects of dietary Cit supplementation revealed in this study are warranted. In conclusion, the novel results of this study revealed that dietary supplementation of unencapsulated L-Cit increased mean daily milk production in dairy goats and elicited compositional changes in the milk produced.

AA- Amino acid

Arg- Arginine

NO- Nitric oxide

Cit- Citrulline

SNF- Soluble non-fat

HPLC- High performance liquid chromatography

OPA- o-phthaldialdehyde

Ethics approval

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

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are presented and available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was supported by funding from Texas A&M AgriLife Research and Prairie View A&M University International Center for Goat Research.

Authors' contributions

The animal experimentation was planned and executed by Arianna N. Lopez, Makenzie G. Newton, Scott Horner, Fuller W. Bazer and William Foxworth. Sample analyses were performed by Arianna N. Lopez, Makenzie G. Newton, Scott Horner, Claire Stenhouse, Erin Connolly, and Karina L Hissen. Data interpretation was performed by Arianna N. Lopez. The first draft of the manuscript was written by Arianna N. Lopez and FWB and edited by William Foxworth, Guoyao Wu and Makenzie Newton, Claire Stenhouse, Erin Connolly and Karina L Hissen.

Acknowledgements

The contributions of graduate students of Texas A&M University and staff or Prairie View A&M University’s International Center for Goat Research are gratefully acknowledged.

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Dietary Citrulline Supplementation Enhances Milk Production in Lactating Dairy Goats

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Abstract

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Background

Materials and methods

Animals

Experimental Design and Sample Collection

Compositional Analyses

Analysis of Amino Acids

Statistical Analyses

Results

Effects of Cit Supplementation, Litter Size, Parity, and Sire on Milk Production

Comparison of milk production across days of lactation

Compositional analysis of milk samples

Concentrations of amino acids in serum

Discussion

Conclusions

Abbreviations

Declarations

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Availability of data and materials

Competing interests

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Authors' contributions

Acknowledgements

References

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