Primary human cells (HSVEpiC; 4460, ScienCell Research Laboratories, Carlsbad, CA, USA) were purchased from a commercial vendor. Densitometry analysis was performed using the ImageJ Gel Analysis tool, and the background of the gel was also removed individually for each band. Infection efficiency, calculated based on the number of GFP-positive cells using an APX100 Digital Imaging System, ranged between 70% and 90%. 100 ng/ml of testosterone was added to all procedures for the testosterone-treated group. Lentivirally transduced cells were prepared according to the manufacturer’s protocol. The sperm pellets were resuspended in HTF medium with or without an LM that included 2 μg/ml arachidonic acid, 10 μg/ml linoleic acid, 10 μg/ml linolenic acid, 10 μg/ml myristic acid, 10 μg/ml oleic acid, 10 μg/ml palmitic acid, and 10 μg/ml stearic acid (L0288, Sigma-Aldrich). Sperm recovered from the cauda epididymis of male mice were dispersed in 500 µl of mHTF without BSA and centrifuged at 300 × g for 3 min to wash out epididymis-derived factors. When taken twice on a daily basis, this product can help ensure that one’s testosterone levels keep constant. This product is also recommended for men who engage in fitness training or bodybuilding and would desire to see an increase in their testosterone levels. This is a testosterone booster that helps address the falling levels of testosterone in men. It is important to remember that testosterone is the main male hormone in the body. It is normal for testosterone levels to recede as one grows past their prime and into the later years of life. Alpha T is a male enhancement supplement that acts as a testosterone booster. The expression of genes involved in glucose catabolism and anabolism in the in vitro culture system of seminal vesicle epithelial cells was analyzed by qPCR (Figure 5). RNA sequencing was performed using RNA extracted from the seminal vesicle epithelial cells cultured with or without 100 ng/ml testosterone. DEG (differentially expressed genes) analysis revealed that a total of 23,459 genes were identified, including 997 upregulated genes and 3463 downregulated genes in seminal vesicle epithelial cells by testosterone relative to control (Figure 3A). However, in mice treated with continuous administration of flutamide, seminal vesicle epithelial cells were multilayered, and the strong accumulation of androgen receptors in the nucleus was not observed (Figure 2A). To understand the difference between seminal vesicles that secrete factors for enhancing sperm linear motility and those that are impaired in this function, we performed histological analysis of seminal vesicles recovered from mice treated and non-treated with flutamide. The final protein concentration of prostate and seminal vesicle secretion was adjusted to 10 mg/mL. Cauda epididymis sperm recovered from male mouse were dispersed in 500 µL of mHTF without BSA drops and centrifuged at 300 × g, 3 min to wash out epididymis-derived factors. Vehicle-treated mice received 100 µL of corn oil subcutaneously for 7 days. 8- to 16-week-old male or female mice were used for the experiments. The objective of this study was to elucidate the mechanisms of sperm plasma synthesis that enhance sperm fertility. Artificial seminal fluid was prepared in HTF medium to a final concentration of 10 mg/mL of collected control or frutamide-derived seminal vesicle secretions. CD1 female mice were injected intraperitoneally with 8 IU eCG to stimulate follicle development and used as recipients for artificial insemination 48 hours later. After 1 hour of incubation in the presence or absence of oleic acid (10 nM), ATP in sperm was visualized by BioTracker ATP-Red (SCT045, Sigma-Aldrich). 100 ng/mL of testosterone was added to all procedures for the testosterone-treated group. Lentivirally transduced cells were prepared according to manufacture protocol. To detect the fluorescence intensity of each sperm sample using flow cytometry, sperm collected from the epididymis were incubated with 1, 10 or 100 μM of fluorescently labeled oleic acid (F-OA-01, NANOCS, New York, NY, USA). Chronic stress raises cortisol levels, which directly suppresses testosterone production and can slow or halt sperm production. Recent studies suggest it can improve both testosterone levels and sperm quality. High intake of soy products may reduce testosterone levels and, consequently, sperm production. On the other hand, trans fatty acids (commonly found in processed foods, margarine, and fast food) are linked to lower sperm count and impaired sperm development. A 2025 analysis of nine randomized controlled trials found that CoQ10 supplementation increased sperm concentration by approximately 10 million sperm per milliliter in men with idiopathic infertility. Meta-analysis indicates that mobile phone exposure affects sperm quality negatively. Tobacco smoking lowers sperm quality, perhaps by decreased ability to attach to hyaluronan on the egg cell. Collecting the sperm at home gives a higher sperm concentration, sperm count, and motility, particularly if the sperm is collected via sexual intercourse. Semen samples obtained via sexual intercourse contain 70–120% more sperm, with sperm having a slightly higher motility and slightly more normal morphology, compared with semen samples obtained via masturbation. Prolonged cycling increases scrotal temperature and puts pressure on the perineum, both of which negatively affect sperm production. Biking more than five hours a week may lower your sperm count. Baseball, tennis, running, and swimming are all excellent options. Sperm take approximately 74 days to fully mature, so any changes you make today will take about three months to show up on a semen analysis. Research shows that sperm counts in Western countries have dropped by nearly 60% since the 1970s, and the decline appears to be accelerating. If you are trying to conceive or considering becoming a sperm donor, knowing how to increase sperm count can make a real difference. Understandably, replacing testosterone through therapeutic supplementation is a big decision with potential long-lasting impacts, both positive and negative.
