Enhancing the post-ruminal supply of arginine (Arg), a semi-essential amino acid (AA), elicits positive effects on milk production. Our objective was to determine the effects of Arg infusion on milk production parameters and aspects of nitrogen (N) absorption and utilization in lactating dairy cows. Six lactating Chinese Holstein cows of similar body weight (508 ± 14 kg), body condition score (3.0 ± 0), parity (4.0 ± 0), milk yield (30.6 ± 1.8 kg) and days in milk (20 ± 2 d) were randomly assigned to 3 treatments in a replicated 3 × 3 Latin square design with 21 d for each period (1 week for infusion and 2 weeks for washout). Treatments were 1) Control: saline; 2) Arg group: saline + 9.42 g/L L-Arg; 3) Alanine (Ala) group: saline + 19.31 g/L L-Ala (iso-nitrogenous to the Arg group). Milk production and composition, dry matter intake, apparent absorption of N, profiles of amino acids (AA) in blood, urea N in urine, milk, and blood, and gene expression of AA transporters were determined. Compared with the Control or Ala group, the infusion of Arg led to greater expression of AA transporters (SLC7A2 and SLC7A8) and apparent uptake of free AA in the mammary gland, and was accompanied by greater milk yield, milk protein yield and milk efficiency (calculated by dividing milk yield over feed intake), together with lower concentration of urea N [regarded as an indicator of N utilization efficiency (NUE)] in blood and milk. Furthermore, in the cows infused with Arg, the NUE was higher and the concentration of urea N in urine was lower than those in the Ala group, although no differences were detected in NUE and urea N in urine between the Control and Arg group. The infusion of Ala had no effect on those indices compared with the Control. Overall, enhancing the post-ruminal supply of Arg via the jugular vein had a positive effect on the synthesis of milk protein at least in part by increasing gene expression of some AA transporters and uptake of free AA by mammary gland.

A previous in vitro study revealed that Arg elicits positive effects on casein synthesis through alterations of the Arg-ornithine pathway in bovine mammary epithelial cells. The main purpose of this work was to determine the effects of arginase inhibition using Nω-hydroxy-nor-l-arginine (nor-NOHA) on milk protein synthesis in vivo. Six healthy Chinese Holstein cows with similar body weight (550.0 ± 20 kg; means ± standard deviation), parity (4), body condition score (3.0), milk yield (21.0 ± 1.0 kg), and days in milk (80 ± 2) were selected and randomly assigned to 3 treatments in a replicated 3 × 3 Latin square design with 22 d for each period (7 d for infusion and 15 d for washout). The treatments were (1) control: saline infusion; (2) nor-NOHA: infusion of 125 mg/L of nor-NOHA; (3) nor-NOHA + Arg: infusion of 125 mg/L of nor-NOHA with 9.42 g/L of Arg. The activity of enzymes related to Arg metabolism, milk protein synthesis, and expression of AA transporters was determined. The infusion of nor-NOHA decreased the activity of arginase but had no effect on the activity of ornithine decarboxylase and nitric oxide synthase in serum, and these responses were the same at the gene expression level in mammary gland. In addition, the infusion of nor-NOHA also reduced protein and fat synthesis in milk but had no effect on milk yield. When Arg was infused with nor-NOHA, the activity of total arginase, ornithine decarboxylase, and nitric oxide synthase, and the concentration of casein, protein, and fat in milk did not change compared with the nor-NOHA group, but the milk protein yield, the expression of some Arg transporters (SLC7A5 and SLC7A8), and milk yield increased. Overall, results verified previous in vitro findings indicating that synthesis of casein protein is closely regulated by the Arg-ornithine pathway in bovine mammary gland.

This study was designed to determine the effects of dietary arginine on development and proliferation in rat mammary tissue through changes in miRNA profiles. Twelve pregnant Wistar rats were allocated randomly to two groups. A basal diet containing arginine or the control diet containing glutamate on an equal nitrogen basis as the arginine supplemented diet were used. The experiment included a pre-experimental period of four days before parturition and an experimental period of 17 days after parturition. Mammary tissue was collected for histology, RNA extraction and high-throughput sequencing analysis. The greater mammary acinar area indicated that arginine supplementation enhanced mammary tissue development (p < 0.01). MicroRNA profiling indicated that seven miRNA (miR-206-3p, miR-133a-5p, miR-133b-3p, miR-1-3p, miR-133a-3p, miR-1b and miR-486) were differentially expressed in response to Arginine when compared with the glutamate-based control group. In silico gene ontology enrichment and KEGG pathway analysis revealed between 240 and 535 putative target genes among the miRNA. Further verification by qPCR revealed concordance with the differential expression from the sequencing results: 17 of 28 target genes were differentially expressed (15 were highly expressed in arginine and 2 in control) and 11 target genes did not have significant difference in expression. In conclusion, our study suggests that arginine may potentially regulate the development of rat mammary glands through regulating miRNAs.

Transformation of Arg to nitric oxide and ornithine (Arg-Orn) constitutes the main route of Arg metabolism in mammals. The primary objective of this work was to determine the effects of inhibiting the Arg-Orn pathway via Nω-hydroxy-nor-l-arginine (nor-NOHA) on health of lactating cows. Furthermore, we also explored the effect of Arg-Orn inhibition on the efficiency of nitrogen utilization to find support for previous research that showed the inhibition of Arg-Orn inhibited milk protein synthesis. Six healthy Chinese Holstein cows of similar body weight (550.0 ± 20 kg), parity (4.0 ± 0), body condition score (3.0 ± 0), milk yield (21.0 ± 1.0 kg), and days in milk (80 ± 2 d) were selected and randomly assigned to 3 treatments in a replicated 3 × 3 Latin square design with 22 d for each period (7 d for infusion and 15 d for washout). The treatments were (1) saline infusion (control); (2) infusion of 125 mg/L of nor-NOHA; and (3) infusion of 125 mg/L of nor-NOHA with 9.42 g/L of Arg. Dry matter intake, apparent digestibility of nutrients, urinary N, N in milk, and blood indices of metabolism and immune function were determined. Compared with the control, the infusion of nor-NOHA had no effect on the concentrations of cholesterol, high-density lipoproteins, IgA, IL-1β, tumor necrosis factor-α, and alanine transaminase. In addition, the dry matter intake, apparent digestibility of N, and the concentration of milk protein N in the Nor-NOHA did not differ from the control; however, the infusion of nor-NOHA and Arg resulted in greater concentrations of high-density lipoprotein, IgA, IL-1β, and tumor necrosis factor-α, and lower concentrations of cholesterol in serum compared with the control. Moreover, the addition of Arg to cows infused with nor-NOHA increased the concentration of nitrate (the indicator of nitric oxide) in serum and was associated with greater milk protein N production due to greater milk yield compared with those infused with nor-NOHA. Overall, the results indicated important roles of Arg in immunity and mammary N utilization, whereas a minor role of the Arg-Orn pathway in these physiologic processes was found.

The mechanism of sex determination in chickens, especially the molecular mechanism of female ovarian development, has not yet been fully elucidated. Previous studies have shown that RSPO1, which is associated with ovarian development in mammals, might have a conserved role in chickens. In this study, we systematically investigated the spatiotemporal expression pattern of RSPO1 in various tissues, especially gonads, of male and female chicken embryos using qPCR and Western blotting, and we explored its correlation with the expression of key genes in the estrogen pathway using drug treatment or gene overexpression in vivo and in vitro. Our results reveal that RSPO1 was widely expressed in all examined tissues of chicken embryos, showing a female bias in gonadal tissues at both the mRNA and protein levels. Surprisingly, RSPO1 was not differentially expressed between male and female gonadal cells with fadrozole-induced estrogen pathway blockades, and furthermore, estradiol-induced estrogen stimulation altered the expression of RSPO1. In addition, overexpression of RSPO1 in gonadal cells induced the mRNA expression of its downstream target genes, Wnt family member 4 (WNT4) and Catenin beta 1 (CTNNB1), and that of estrogen receptor α (ERα), an estrogen pathway gene. In summary, this study provided new evidence for elucidating the role of RSPO1 in ovarian development in poultry.