Dietary Citrulline Supplementation Enhances Milk Production in Lactating Dairy Goats

Dietary Citrulline Supplementation Enhances Milk Production in Lactating Dairy Goats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dietary Citrulline Supplementation Enhances Milk Production in Lactating Dairy Goats Arianna Lopez, Makenzie Newton, Claire Stenhouse, Erin Connolly, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4426614/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background 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. Materials and methods 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 HCLO 4 and vortexed. The acidified sample was then neutralized with 50 µL of 2 mol/L K 2 CO 3 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. Results 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 \(\:\pm\:\:31\) Discussion 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, Β 0 ,B 0,+ , and b 0,+ transporters that depend on tissue and cell type [ 34 ]. Studies with sheep and cattle found no detectable uptake of 14 C-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. Conclusions 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. Abbreviations AA- Amino acid Arg- Arginine NO- Nitric oxide Cit- Citrulline SNF- Soluble non-fat HPLC- High performance liquid chromatography OPA- o -phthaldialdehyde Declarations 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. References Šalavardić ŽK, Potočnik K, Zvonko A, Novoselec J. Milk production traits from alpine breed of goats in Croatia and Slovenia. Bulgarian J Agri Sci. 2015; 21:1064-1068. Zeng SS, Escobar EN, Popham T. Daily variations in somatic cell count, composition, and production of Alpine goat milk. Small Rum Research. 1997; 26:253-260 Park YW. 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Wu G, Bazer FW, Satterfield MC, Gilbreath KR, Posey EA, Sun Y. L-Arginine nutrition and metabolism in ruminants. Adv Exp Med Biol. 2022; 1354:177–206. Bérard J, Bee G. Effects of dietary l-arginine supplementation to gilts during early gestation on fetal survival, growth and myofiber formation. Animal. 2010; 4:1680–1687. Tapiero H, Mathé G, Couvreur P, Tew KD. I. Arginine. Biomed Pharmacother. 2002; 56:439–445. Breuillard C, Cynober L, Moinard C. Citrulline and nitrogen homeostasis: an overview. Amino Acids. 2015; 47:685–691. Wu G. Synthesis of citrulline and arginine from proline in enterocytes of postnatal pigs. Am J Physiol. 1997; 272:1. Aguayo E, Martínez-Sánchez A, Fernández-Lobato B, Alacid F. L-citrulline: a non-essential amino acid with important roles in human health. Applied Sci. 2021; 11:7. Wu G, Bazer FW, Davis TA, Kim SW, Li P, Marc Rhoads J, et al. Arginine metabolism and nutrition in growth, health and disease. Amino Acids 2008; 37:153–168. Council NR. Nutrient Requirements of Small Ruminants: Sheep, Goats, Cervids, and New World Camelids. National Ac. Press. 2007;1. Halloran KM, Hoskins EC, Stenhouse C, Moses RM, Dunlap KA, Satterfield MC, et al. Pre-implantation exogenous progesterone and pregnancy in sheep. II. effects on fetal-placental development and nutrient transporters in late pregnancy. J Anim Sci Biotechnol. 2021; 12:1–20. Wu G, Davis PK, Flynn NE, Knabe DA, Davidson JT. Endogenous synthesis of arginine plays an important role in maintaining arginine homeostasis in postweaning growing pigs. J Nutr. 1997; 127:2342–2349. Lee U, Garcia TP, Carroll RJ, Gilbreath KR, Wu G. Analysis of repeated measures data in nutrition research. Front Biosci. 2019; 24:1377-1389. Mateo RD, Wu G, Bazer FW, Park JC, Shinzato I, Sung WK. Dietary l-arginine supplementation enhances the reproductive performance of gilts. J Nutr. 2007; 137:652–656. Li X, Bazer FW, Johnson GA, Burghardt RC, Wu G. Dietary supplementation with L-citrulline improves placental angiogenesis and embryonic survival in gilts. Exp Biol Med. 2023; 248:702–711. Wu G, Bazer FW, Satterfield MC, Gilbreath KR, Posey EA, Sun Y. L-arginine nutrition and metabolism in ruminants. Adv Exp Med Biol. 2022; 1354:177–206. McCarthy N, Brougham BJ, Swinbourne AM, Weaver AC, Kelly JM, Gatford KL, et al. Maternal oral supplementation with citrulline increases plasma citrulline but not arginine in pregnant Merino ewes and neonatal lambs. Anim Prod Sci. 2022; 62:521–528. Wu G. Amino acids - biochemistry and nutrition. CRC Press. 2022; 96:1–12. Bahri S, Zerrouk N, Aussel C, Moinard C, Crenn P, Curis E, et al. Citrulline: from metabolism to therapeutic use. Nutrition. 2013; 29:479–484. Ma Y, Zhao G, Wang C, An M, Ma C, Liu Z, et al. Corrigendum to: effects of supplementation with different concentrations of L-citrulline on the plasma amino acid concentration, reproductive hormone concentrations, antioxidant capacity, and reproductive performance of Hu ewes. Anim Prod Sci. 2023; 63:924–924. Hayden TJ, Thomas CR, Forsyth IA. Effect of number of young born (litter size) on milk yield of goats: role for placental lactogen. J Dairy Sci. 1979; 62:53–57. Novotná L, Kuchtík J, Sbreve K[, Ustová ], Zapletal D, Filip R, et al. Effects of lactation stage and parity on milk yield, composition and properties of organic sheep milk. J Appl Anim Res. 2009; 36:71–76. Moya J, Wilcox CJ, Littell RC, Thatcher WW. Effects of sire of fetus upon subsequent milk production and reproduction of jersey cows. J Dairy Sci. 1989; 72:1012–1019. 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University","correspondingAuthor":false,"prefix":"","firstName":"Makenzie","middleName":"","lastName":"Newton","suffix":""},{"id":348678637,"identity":"56b5bb50-7288-4250-a7b4-eb1ed30c2034","order_by":2,"name":"Claire Stenhouse","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Claire","middleName":"","lastName":"Stenhouse","suffix":""},{"id":348678638,"identity":"e668ab15-d136-47b5-b61c-221ba43c57a2","order_by":3,"name":"Erin Connolly","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Erin","middleName":"","lastName":"Connolly","suffix":""},{"id":348678639,"identity":"7ad10acf-f4e0-4119-9bcb-86dbde5d75a4","order_by":4,"name":"Karina Hissen","email":"","orcid":"","institution":"Texas A\u0026M 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version.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4426614/v1/429adbfb404c4791cf03fdd7.png"},{"id":67167777,"identity":"9aeea6b2-750b-4777-a181-34b8e78d534b","added_by":"auto","created_at":"2024-10-22 02:12:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1175204,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4426614/v1/0fcce2c3-1b1e-43ad-b868-e72059eeae62.pdf"}],"financialInterests":"","formattedTitle":"Dietary Citrulline Supplementation Enhances Milk Production in Lactating Dairy Goats","fulltext":[{"header":"Background","content":"\u003cp\u003eLactational 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 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. 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) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The milk goat industry is a vital agricultural enterprise in many countries, including the United States, the Mediterranean region, and China [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The composition of goat milk differs from that of milk from cows and women in having greater digestibility [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. 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\u0026rsquo;s milk [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eNutritional 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 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Ruminants such as goats, cows, and sheep have a rumen, reticulum, omasum, and abomasum in comparison to the simple stomach in monogastric species [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The rumen is lined by papillae and inhabited by bacteria (95% of the microbial population), archea, fungi, and protozoa [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These microbes have multiple roles in digestion of proteins, degradation of amino acids (AA) degradation, and protein synthesis by microbes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Protein metabolism is the direct result of metabolic activity of ruminal microbes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It has been suggested that AA uptake by ruminal microbes may be the limiting factor in protein degradation in the rumen [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Once AAs are taken up by ruminal microbes, they are incorporated into protein or are broken down into ammonia for synthesis of AA [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In ruminant nutrition, a long-standing view is that all dietary AAs, including Arg and glutamine, undergo extensive degradation by ruminal microbes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, it has been discovered that citrulline (Cit) is not degraded by the ruminal microbes of ruminants including sheep and cattle [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Arg is a common substrate for synthesis of nitric oxide (NO), polyamines and creatine [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Arg metabolism is highly compartmentalized and requires inter-organ cooperation as not all of the enzymes are expressed in all cells and tissues [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Cit has important roles for regulation of nitrogen homeostasis, immune responses, and the cardiovascular system [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The intestinal-renal axis allows synthesis of Cit from glutamate and glutamine in the small intestine and its conversion to Arg in the kidneys [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Glutamine is first converted into glutamate by glutaminase, which is then catabolized into Cit [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Subsequently, enterocytes release Cit into the portal circulation for transport to the kidneys where it is converted into arginosuccinate [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Arginosuccinate is then converted into fumarate and then Arg via argininosuccinate lyase [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. 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.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eLactating alpine dairy goats were utilized to study the effects of dietary supplementation of L-citrulline (N\u0026thinsp;=\u0026thinsp;47). All goats were fed to meet National Research Council requirements and assigned randomly to either the control treatment group (CON, n\u0026thinsp;=\u0026thinsp;24), or the L-citrulline treatment group (CIT, n\u0026thinsp;=\u0026thinsp;23) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Design and Sample Collection\u003c/h2\u003e \u003cp\u003eOn 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\u0026deg;C overnight. Blood samples were centrifuged (Eppendorf centrifuge 5920R Hamburg, Germany) at 5\u0026deg;C for 18 min at 2,600 x \u003cem\u003eg\u003c/em\u003e. Serum was harvested and stored at -20\u0026deg;C until analyzed for concentrations of AA using high pressure liquid chromatography (HPLC) analyses [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCompositional Analyses\u003c/h2\u003e \u003cp\u003eMilk 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 \u0026amp; Pederson Int.) for determination of butter fat, protein, lactose, and solids not fat (SNF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Amino Acids\u003c/h2\u003e \u003cp\u003eEffects of dietary supplementation of Cit on concentrations of amino acids in serum were determined using HPLC and modified procedures described previously [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Each serum sample (100 \u0026micro;L) was acidified with 100 \u0026micro;L of 1.5 mol/L HCLO\u003csub\u003e4\u003c/sub\u003e and vortexed. The acidified sample was then neutralized with 50 \u0026micro;L of 2 mol/L K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e and vortexed. The neutralized sample was then centrifuged for 3 min at 10,000 x \u003cem\u003eg\u003c/em\u003e in an Eppendorf centrifuge (Eppendorf centrifuge 5920R). The neutralized supernatant was then collected and diluted 10-fold before analyzed by HPLC using precolumn derivatization \u003cem\u003eo\u003c/em\u003e-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 \u0026micro;L 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 \u0026micro;L of 1.2% benzoic acid (in 40 mmol/L sodium borate, pH 9.5), and 100 \u0026micro;L of sample. The assay mixture was derivatized in an autosampler (model 712 WISP, Waters, Milford, MA) with 30 mmol/L OPA, and 15 \u0026micro;L 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eStatistical 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 \u003cem\u003eP\u003c/em\u003e value of \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;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 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.248). To compare means on a pair-wise comparison basis for data normally distributed, the each-pair student\u0026rsquo;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 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This method considers Day 0 of lactation baseline concentrations as a covariate and uses the difference from baseline as a method to normalize data [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In all methods of statistical analyses, results were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;0.05, trending towards significance at 0.05\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.10, and not significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e\u0026thinsp;0.10.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Cit Supplementation, Litter Size, Parity, and Sire on Milk Production\u003c/h2\u003e \u003cp\u003eA one-way ANOVA revealed that CIT does had greater milk production than CON does (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04) (Fig.\u0026nbsp;1), and that the litter size of does influenced milk production (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) (Fig.\u0026nbsp;2). On a pair-wise comparison basis, does producing three kids produced more milk than does producing either 1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) or 2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) kids.\u003c/p\u003e \u003cp\u003eOne-way ANOVA indicated that multiparous does produced more milk than primiparous does (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;3) and sire affected milk production by does (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;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 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.058), but there was no treatment effect among primiparous does.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eComparison of milk production across days of lactation\u003c/h2\u003e \u003cp\u003eMean 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 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;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 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and Day 10 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.041) of lactation. Milk production on Day 20 of lactation was greater than that on Day 0 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) of lactation, and milk yield on Day 10 of lactation was greater than that on Day 0 of lactation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These results are presented in Fig.\u0026nbsp;5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCompositional analysis of milk samples\u003c/h2\u003e \u003cp\u003eMean concentrations of protein in milk collected on Days 10, 20, and 40 of lactation were greater in the milk from CIT than CON does (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024), and there was a trend (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;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 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;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 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;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.\u0026nbsp;6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eConcentrations of amino acids in serum\u003c/h2\u003e \u003cp\u003eThere was no effect (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.10) of dietary Cit supplementation on concentrations of Arg, Cit, Orn, and alanine in serum (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 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 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.10) of Cit supplementation and a summary of these concentrations are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConcentrations of citrulline, arginine, ornithine, and alanine in serum of lactating goats on Days 0 and 30 of lactation. Mean values (nmol/mL) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:SD\\:\\)\u003c/span\u003e\u003c/span\u003eat a 10X dilution factor. The numbers of does are 24 and 23 for the Control and Citrulline fed groups, respectively. \u003cb\u003eDay Control (alanine supplementation) Citrulline supplementation\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eArg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOrn\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAla\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eArg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eOrn\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eAla\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:49\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e211 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:42\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:26\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e182 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:46\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e92 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:42\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e213 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:29\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e31 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:11\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e219 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:65\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e184 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:68\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e217 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:60\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:38\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e220 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:58\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e150 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:53\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e219 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:29\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e65 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:20\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e239 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:73\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConcentrations (nmol/mL \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:SD)\\)\u003c/span\u003e\u003c/span\u003e 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. \u003cb\u003eDay Control (alanine supplementation) Citrulline supplementation\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAsp\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlu\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAsn\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAsp\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGlu\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAsn\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSer\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.8 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:1.7\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e120 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:36\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:17\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:36\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.7 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:1.8\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e131 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:39\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e38 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:8\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e62 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:24\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.6 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:1.9\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e140 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:35\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:18\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e140 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:39\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.8 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:1.9\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e138 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:42\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e53 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:18\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e111 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:31\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eGln\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eHis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eGly\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eThr\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eGln\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eHis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eGly\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eThr\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e332 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:77\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:14\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e483\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:162\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:21\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e329 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:53\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e73 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:13\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e537 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:163\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e59 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:23\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e349 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:67\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:19\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:66\\pm\\:167\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:23\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e375 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:104\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e65 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:11\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e691\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:272\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e55 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:29\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eβ-Ala\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eTau\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eTyr\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eTrp\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eβ-Ala\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eTau\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eTyr\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eTrp\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:0.2\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e163 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:37\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:25\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:13\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.8 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:0.8\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e199 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:49\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e49 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:8\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e33 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:9\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:0.6\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e149 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:53\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:25\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:16\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.7 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:0.9\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e134 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:21\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e55 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:19\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e38 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:9\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMet\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eVal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003ePhe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eIle\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eMet\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eVal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003ePhe\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eIle\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:13\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e165 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:88\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:23\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e117 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:58\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:11\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e178 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:36\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:10\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e106 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:40\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:16\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e178 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:35\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:11\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e140 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:71\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e38 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:9\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e172 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:54\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e43 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:12\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e116 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:36\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eLeu\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eLys\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eLeu\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eLys\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e161 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:64\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e138 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:49\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e151 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:62\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e122 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:27\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e140 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:65\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e156 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:71\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e169 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:43\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e145 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:31\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis 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 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similar to swine, the enterocytes of adult cattle have increased rates of intestinal synthesis of Cit and Arg from glutamate and Pro during pregnancy [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Also, dietary supplementation with Cit enhances endogenous synthesis of Arg in ruminants [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In ruminants, Cit derived from the small intestine is locally converted into Arg via the intestinal-renal axis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In fed sheep, the small intestine releases Cit into the vasculature and the kidneys uptake approximately 1.41mmol/h [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Subsequently, the kidneys release Arg at approximately 1.46 mmol/h [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn 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 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Cit transport is tissue- and cell-specific, and includes AA transporters, common transport system with Arg, L, N, Β\u003csup\u003e0\u003c/sup\u003e,B\u003csup\u003e0,+\u003c/sup\u003e, and b\u003csup\u003e0,+\u003c/sup\u003e transporters that depend on tissue and cell type [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Studies with sheep and cattle found no detectable uptake of \u003csup\u003e14\u003c/sup\u003eC-labeled Cit by ruminal microbes indicating that Cit may be supplemented without encapsulation and will not be degraded by ruminal microbes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Ewes fed a diet supplemented with Cit had greater concentrations of Cit, Orn, and Arg that increased linearly with increasing doses of Cit supplementation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Concentrations of NO in serum were also 11.25% greater in the Cit supplemented ewes compared to ewes fed a control diet [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e \u003cp\u003eThe effect of litter size on milk production is positively correlated as expected [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. That study also revealed a significant effect of litter size on mean daily milk production, which aligns with other results in the literature [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. 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.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe 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.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAA- Amino acid\u003c/p\u003e\n\u003cp\u003eArg- Arginine\u003c/p\u003e\n\u003cp\u003eNO- Nitric oxide\u003c/p\u003e\n\u003cp\u003eCit- \u0026nbsp;Citrulline\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSNF- Soluble non-fat\u003c/p\u003e\n\u003cp\u003eHPLC- High performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eOPA-\u0026nbsp;\u003cem\u003eo\u003c/em\u003e-phthaldialdehyde\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eAll 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\u0026amp;M University.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are presented and available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was supported by funding from Texas A\u0026amp;M AgriLife Research and Prairie View A\u0026amp;M University International Center for Goat Research.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe contributions of graduate students of Texas A\u0026amp;M University and staff or Prairie View A\u0026amp;M University\u0026rsquo;s International Center for Goat Research are gratefully acknowledged.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u0026Scaron;alavardić ŽK, Potočnik K, Zvonko A, Novoselec J. Milk production traits from alpine breed of goats in Croatia and Slovenia. 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L-arginine nutrition and metabolism in ruminants. Adv Exp Med Biol. 2022; 1354:177\u0026ndash;206. \u003c/li\u003e\n\u003cli\u003eMcCarthy N, Brougham BJ, Swinbourne AM, Weaver AC, Kelly JM, Gatford KL, et al. Maternal oral supplementation with citrulline increases plasma citrulline but not arginine in pregnant Merino ewes and neonatal lambs. Anim Prod Sci. 2022; 62:521\u0026ndash;528. \u003c/li\u003e\n\u003cli\u003eWu G. Amino acids - biochemistry and nutrition. CRC Press. 2022; 96:1\u0026ndash;12. \u003c/li\u003e\n\u003cli\u003eBahri S, Zerrouk N, Aussel C, Moinard C, Crenn P, Curis E, et al. Citrulline: from metabolism to therapeutic use. Nutrition. 2013; 29:479\u0026ndash;484. \u003c/li\u003e\n\u003cli\u003eMa Y, Zhao G, Wang C, An M, Ma C, Liu Z, et al. Corrigendum to: effects of supplementation with different concentrations of L-citrulline on the plasma amino acid concentration, reproductive hormone concentrations, antioxidant capacity, and reproductive performance of Hu ewes. Anim Prod Sci. 2023; 63:924\u0026ndash;924. \u003c/li\u003e\n\u003cli\u003eHayden TJ, Thomas CR, Forsyth IA. Effect of number of young born (litter size) on milk yield of goats: role for placental lactogen. J Dairy Sci. 1979; 62:53\u0026ndash;57. \u003c/li\u003e\n\u003cli\u003eNovotn\u0026aacute; L, Kucht\u0026iacute;k J, Sbreve K[, Ustov\u0026aacute; ], Zapletal D, Filip R, et al. Effects of lactation stage and parity on milk yield, composition and properties of organic sheep milk. J Appl Anim Res. 2009; 36:71\u0026ndash;76. \u003c/li\u003e\n\u003cli\u003eMoya J, Wilcox CJ, Littell RC, Thatcher WW. Effects of sire of fetus upon subsequent milk production and reproduction of jersey cows. J Dairy Sci. 1989; 72:1012\u0026ndash;1019. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lactation, Milk Composition, Arginine, Citrulline, Dairy Goats","lastPublishedDoi":"10.21203/rs.3.rs-4426614/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4426614/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eNutrition, 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eCIT-treated does had greater mean daily milk production (\u003cem\u003eP\u003c/em\u003e = 0.0332) and there was an effect of day of lactation on mean daily milk production (P \u0026lt; 0.0001). Does producing three kids had greater mean daily milk production than does producing one kid (P\u0026lt;0.001). Multiparous does had greater mean daily milk production than primiparous does (P\u0026lt;0.0001), and there was an effect of sire on mean daily milk production (P\u0026lt;0.05). Compositional analyses revealed that Cit supplementation increased soluble-non-fat (SNF) (P= 0.0189) and protein (P=0.0238) in milk.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eDietary 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.\u003c/p\u003e","manuscriptTitle":"Dietary Citrulline Supplementation Enhances Milk Production in Lactating Dairy Goats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-04 05:23:52","doi":"10.21203/rs.3.rs-4426614/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5e3ed7e3-fb84-49bc-9359-9a5c5d74ca7b","owner":[],"postedDate":"October 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-20T01:33:44+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-04 05:23:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4426614","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4426614","identity":"rs-4426614","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}