1- Ph. D student in Poultry Nutrition, Department of Animal Science, Faculty of Animal Science and Food Technology, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran
2- Assistant Professor, Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran
3- Associate Professor, Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran
2- Assistant Professor, Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran
3- Associate Professor, Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran
Abstract: (13 Views)
Introduction and Objective: Reduced fertility at the end of the reproductive cycle of roosters is directly related to structural and compositional changes in sperm membrane phospholipids and a decrease in long-chain unsaturated fatty acids, especially in phosphatidylethanolamine. At the end of the reproductive period, the ratio of phosphatidylcholine increases, and phosphatidylethanolamine and phosphatidylserine decrease. Hence, in improving sperm quality in older roosters, nutrition has a significant influence on sperm quality and quantity. Therefore, enriching the diet with multifunctional compounds that maintain this for a longer period is beneficial to maximize economic benefits. Adding the amino acid serine and choline supplements to the diet of older roosters can be an effective strategy to increase sperm quality. Serine is traditionally considered a non-essential amino acid; however, it plays vital roles in protein synthesis and cell signal transduction within the body. Serine can reduce oxidative stress and increase the activity of antioxidant enzymes. Increased oxidative stress causes damage to testicular cells in birds, resulting in reduced testicular weight and sperm production. Choline supplementation is a nutrient with essential metabolic functions and is a key component of membrane phospholipids. It has also been linked to fertility, as choline supplementation can enhance the expression of genes involved in spermatogenesis. Choline plays a crucial role in synthesizing cell membrane phospholipids, particularly in sperm cells. Key genes involved in the biosynthesis pathways for phosphatidylcholine and serine include choline phosphotransferase 1 (CHPT1) and phosphatidylserine synthase 1 (PTDSS1). The enzyme choline phosphotransferase 1 catalyzes the transfer of a phosphocholine group from CDP-choline to diacylglycerol. This reaction produces phosphatidylcholine, which is one of the most essential phospholipids in cell membranes. Phosphatidylcholine plays a crucial role in maintaining the structure and function of cells. Phosphatidylcholine is a key component of cell membranes and serves as a precursor for acetylcholine. It also plays important roles in metabolic functions and cell signaling. The PTDSS1 gene encodes an enzyme called phosphatidylserine synthase 1, which is essential for the production of phosphatidylserine, a crucial phospholipid found in cell membranes. This enzyme is situated in the endoplasmic reticulum membrane and catalyzes the conversion of either phosphatidylcholine or phosphatidylethanolamine into phosphatidylserine. Research has indicated that aging is associated with an increase in phosphatidylcholine levels and a decrease in phosphatidylserine levels, which may contribute to reduced fertility. Thus, the present study was conducted to investigate the effect of adding the amino acid serine and choline supplementation to the diet of old broiler breeder roosters on the expression of choline phosphotransferase 1 (CHPT1) and phosphatidylserine synthase 1 (PTDSS1) genes, which are important and effective in the phosphatidylcholine and serine biosynthesis pathway.
Material and Methods: An experiment was conducted involving 36 Ross 308 broiler breeder roosters, approximately 65 weeks old. The study utilized a completely randomized design (CRD) with a 3×2 factorial arrangement of the treatments. This setup included six treatments and six replications, and the experiment lasted for 10 weeks. The experimental treatments included: 1) Basal diet contains 0% of the amino acid serine + 0% of choline, 2) 0% serine + 1400 mg/kg choline, 3) 0.15% amino acid serine + 0% choline supplement, 4) 0.15% amino acid serine + 1400 mg/kg choline, 5) 0.3% amino acid serine + diet containing 0% choline supplement, and 6) 0.3% amino acid serine + 1400 mg choline supplement. At the end of the experiment, four birds from each treatment were euthanized for gene analysis. A portion of their testicular tissue was promptly removed and transported to the laboratory in liquid nitrogen, where it was stored at -80°C. Following the extraction of total RNA, the quality and quantity of the RNA were assessed using electrophoresis and a Nanodrop device. In order to confirm the specific amplification of the target genes, polymerase chain reaction using designed primers was first performed in a thermal cycler. After that, real-time PCR was performed on the samples with two experimental replicates for the CHPT1, PTDSS1 genes and the beta-actin gene as a reference gene.
Results: Electrophoresis of the polymerase chain reaction products revealed fragments of 127, 109, and 145 base pair for CHPT1, PTDSS1, and β-actin, respectively. The observation of only one peak in the melting curves confirmed the specificity of the designed primers. The analysis of variance results indicated that the addition of the amino acid serine at two concentrations, 0.15% and 0.3%, resulted in a significant decrease in CHTP1 gene expression (P < 0.05). In contrast, the addition of 1400 mg/kg of choline did not significantly affect the expression of this gene (P > 0.05). Also, real-time results showed that adding 0.3% of the amino acid serine and adding 1400 mg/kg of choline alone led to a significant increase in PTDSS1 gene expression (P<0.05), but adding 0.15% of the amino acid serine did not cause a significant change in the expression of this gene (P>0.05). Simultaneous addition of 0.3% of the amino acid serine with 1400 mg/kg of choline resulted in an increase in PTDSS1 gene expression (P<0.05), but no significant effect was observed with the addition of 0.15% of the amino acid serine with choline (P>0.05). Also, the addition of two levels of 0.15 and 0.3% of serine with choline resulted in a decrease in CHTP1 gene expression, and the presence of choline failed to prevent this decrease.
Conclusion: Adding 0.3% of the amino acid serine along with 1400 mg/kg of choline to the diet of old broiler breeder roosters resulted in a decrease in CHPT1 gene expression and an increase in PTDSS1 gene expression. A decrease in CHPT1 gene expression and an increase in PTDDS1 gene expression, respectively, resulted in a decrease in phosphatidylcholine and an increase in phosphatidylserine in the rooster testis. Therefore, it can be concluded that adding serine and choline to the diet could possibly increase sperm quality by increasing phosphatidylserine and decreasing phosphatidylcholine.
Material and Methods: An experiment was conducted involving 36 Ross 308 broiler breeder roosters, approximately 65 weeks old. The study utilized a completely randomized design (CRD) with a 3×2 factorial arrangement of the treatments. This setup included six treatments and six replications, and the experiment lasted for 10 weeks. The experimental treatments included: 1) Basal diet contains 0% of the amino acid serine + 0% of choline, 2) 0% serine + 1400 mg/kg choline, 3) 0.15% amino acid serine + 0% choline supplement, 4) 0.15% amino acid serine + 1400 mg/kg choline, 5) 0.3% amino acid serine + diet containing 0% choline supplement, and 6) 0.3% amino acid serine + 1400 mg choline supplement. At the end of the experiment, four birds from each treatment were euthanized for gene analysis. A portion of their testicular tissue was promptly removed and transported to the laboratory in liquid nitrogen, where it was stored at -80°C. Following the extraction of total RNA, the quality and quantity of the RNA were assessed using electrophoresis and a Nanodrop device. In order to confirm the specific amplification of the target genes, polymerase chain reaction using designed primers was first performed in a thermal cycler. After that, real-time PCR was performed on the samples with two experimental replicates for the CHPT1, PTDSS1 genes and the beta-actin gene as a reference gene.
Results: Electrophoresis of the polymerase chain reaction products revealed fragments of 127, 109, and 145 base pair for CHPT1, PTDSS1, and β-actin, respectively. The observation of only one peak in the melting curves confirmed the specificity of the designed primers. The analysis of variance results indicated that the addition of the amino acid serine at two concentrations, 0.15% and 0.3%, resulted in a significant decrease in CHTP1 gene expression (P < 0.05). In contrast, the addition of 1400 mg/kg of choline did not significantly affect the expression of this gene (P > 0.05). Also, real-time results showed that adding 0.3% of the amino acid serine and adding 1400 mg/kg of choline alone led to a significant increase in PTDSS1 gene expression (P<0.05), but adding 0.15% of the amino acid serine did not cause a significant change in the expression of this gene (P>0.05). Simultaneous addition of 0.3% of the amino acid serine with 1400 mg/kg of choline resulted in an increase in PTDSS1 gene expression (P<0.05), but no significant effect was observed with the addition of 0.15% of the amino acid serine with choline (P>0.05). Also, the addition of two levels of 0.15 and 0.3% of serine with choline resulted in a decrease in CHTP1 gene expression, and the presence of choline failed to prevent this decrease.
Conclusion: Adding 0.3% of the amino acid serine along with 1400 mg/kg of choline to the diet of old broiler breeder roosters resulted in a decrease in CHPT1 gene expression and an increase in PTDSS1 gene expression. A decrease in CHPT1 gene expression and an increase in PTDDS1 gene expression, respectively, resulted in a decrease in phosphatidylcholine and an increase in phosphatidylserine in the rooster testis. Therefore, it can be concluded that adding serine and choline to the diet could possibly increase sperm quality by increasing phosphatidylserine and decreasing phosphatidylcholine.
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