Cam β-NGF Supplementation: A Promising Approach to Preserve Dromedary Camel Sperm Quality During Short-term Storage

1. INTRODUCTION

Cryopreservation of sperm and the development of assisted reproductive technologies are widely used techniques for preserving and supplying sperm, supporting breeding programs, and maintaining genetic diversity in livestock and wildlife. However, dromedary camel sperm is typically regarded as low quality, exhibits high viscosity that complicates handling, dilution, and evaluation, and is not tolerant to freezing and thawing procedures [1, 2]. Besides, attempts to cryopreserve dromedary camel spermatozoa have been met with reduced success. The cryopreservation process begins with dilution with an extender (36°C), followed by a cooling step (4–5°C), the addition of the cryoprotectant, equilibration, and then freezing in liquid nitrogen at −196°C. Each step of this process, along with thermal changes, can cause gamete damage, the extent of which remains unknown. However, it has been reported that using some extenders in the process of artificial insemination affects sperm motility within 12–24 hours of cold storage [3]. The composition of camel seminal plasma appears to be particular. Camelids have higher levels of βeta-Nerve Growth Factor (β-NGF) than other mammals [4]. This protein, a member of the neurotrophin family, plays a crucial role not only in the nervous system but also in other non-nervous systems, such as the reproductive system [5]. In dromedary seminal plasma, Fatnassi et al. [6] detected the presence of the β-NGF named “Cam-β-NGF” with a molecular weight of approximately 14 kDa. This protein induced ovulation at rates similar to GnRH. Nerve growth factor is likely one of the most widely researched neurotrophins [7]. Numerous studies have demonstrated its presence in germinal cells at every developmental phase.

Furthermore, the effects of NGF on reproductive systems and sperm function have recently been assessed in animal models [8-10]. These studies highlight its role in supporting testis formation and development, as well as influencing sperm differentiation, maturation [11, 12], viability [9], motility [13], and DNA integrity in humans [14]. This study aimed to investigate the effect of Cam β-NGF addition on the motility, viability, and membrane integrity of dromedary (Camelus dromedarius) semen during short-term cold storage (24 h at 4 °C).

2. MATERIALS AND METHODS

2.2. Semen Collection and Ejaculate Evaluation

Semen was collected twice a week with a bovine artificial vagina (IMV, France) in the presence of a female camel restrained in sternal recumbency on padded flooring (cushioned position) to prevent injury [16]. The female was used solely to stimulate mounting; no mating or invasive procedures were performed, and all procedures were supervised by a veterinarian. All animal procedures were approved by the Animal Ethics Committee of the National School of Veterinary Medicine (AEC/IACUC, ENMV–Sidi Thabet, Tunisia; approval code CEEA-ENMV 64/22, approved on 26 December 2022). Semen collection was programmed with a 30 min maximal mounting time. After collection, semen volume was measured using a graduated collection tube. Then, 30 min after warming in a water bath at 36°C, mass motility, viability, membrane integrity, and concentration were determined. Mass motility (0–5) was evaluated by observing 50 μL of semen on a pre-warmed slide under a phase-contrast microscope (Nikon E200; Tokyo, Japan) at 200× magnification [17]. The proportion of viable spermatozoa was assessed using eosin-nigrosin staining [18] after counting 200 spermatozoa. Sperm concentration was measured after 90 min of incubation using a Thoma hemocytometer. The total number of spermatozoa was calculated by multiplying the sperm concentration by the volume of the ejaculate (Table 1).

Table 1.

Parameters	Values (Mean ± SD)
Mass Motility	5 ± 0
Viability (%)	77.25 ± 3.91
HOST (%)	74.90 ± 8.73
Concentration (x108)	7.35 ± 4.79
Total number of SPZ (x108)	32.68 ± 21.82

Plasma membrane integrity was tested using a hypo-osmotic swelling test, as described by Ramu and Jeyendran [19]. Briefly, 10 µL of semen was mixed with 100 µL of hypo-osmotic solution (HOS: 1.5 mM trisodium citrate and 1.5 mM fructose) and incubated in the semen/HOS mixture for at least 30 min at 36°C. Subsequently, 10 µL of the solution was deposited onto a pre-warmed slide covered with a coverslip, and sperm were examined under a phase-contrast microscope at 400× magnification. Two hundred spermatozoa per slide were counted from four microscopic fields and expressed as percentages. The swollen spermatozoa were considered normal sperm cells (Fig. 1). Immediately following collection, semen samples were diluted with HBSS (25 mM HEPES, 130 mM NaCl, 5 mM KCl, 0.36 mM NaH₂PO₄, 0.49 mM MgCl₂, and 2.4 mM CaCl₂; pH 7.4, 290 mOsm/kg) at 37°C. Samples were then centrifuged at 800 × g for 10 min at room temperature to discard seminal plasma.

2.4. Seminal Plasma Purification and Isolation of Cam-β-NG

Protein purification was conducted according to the method outlined by Fatnassi et al. [6]. Briefly, at room temperature and with a flow rate of 0.5 mL/min, 2 mL of camel seminal plasma (approximately 40 mg) was separated using fast protein liquid chromatography (FPLC; GE Healthcare Life Sciences, Bjorkgatan, Sweden) with a gel filtration column (SEC, Hi Prep^TM 26/60 Sephacryl^TM S-100, GE Healthcare Life Sciences, Bjorkgatan, Sweden) (Fig. 2). The mobile phase used for elution consisted of phosphate-buffered saline with pH 7.4. This procedure resulted in the separation of two distinct protein peaks, which were subsequently pooled individually and stored at −20 °C until needed. The pool of the major peak (peak 2) was identified [6] as Cam-β-NG for the remainder of the experiment; it was desalted, lyophilized, and preserved at −20 °C until use.

3. RESULTS

The effect of short-term conservation (24 h at 4 °C) on mass motility, viability, and membrane integrity is shown in the corresponding Fig. (3A-C).

After 24 h of storage at 4°C, there was a significant decrease in sperm mass motility (5.0 ± 0.0 vs. 2.65 ± 0.41, respectively; p = 0.0048) (Fig. 3A). The viability of preserved sperm was significantly reduced compared to that of fresh sperm before preservation (77.25 ± 3.91% vs. 61.95 ± 10.93%, respectively; p = 0.0032) (Fig. 3B). Furthermore, membrane integrity was significantly decreased after preservation (Fig. 3C), which was expressed as the percentage of swollen spermatozoa (74.90 ± 8.73% vs. 50.67 ± 10.14%, respectively; p = 0.0006).

After supplementation with Cam-β-NGF, samples were then incubated for a total duration of 60 min at 36°C, with measurements taken at T0 (5 min after the start of incubation), T30, and T60 min to assess the effect of each dose on semen quality parameters.

After 5 min of incubation (T0), motility (Fig. 4A), viability (Fig. 4B), and membrane integrity (Fig. 4C) were found to be statistically unchanged.

After 30 min of incubation, the introduction of 1000 ng/mL and 500 ng/mL NGF in the semen extender exhibited the highest mass motility (p < 0.05) in comparison to other concentrations (100 ng/mL and 10 ng/mL) and the control (Fig. 5A). However, viability at T30 (Fig. 5B) did not show a statistically significant difference between the doses and the control. In contrast, membrane integrity (Fig. 5C) was maintained at T30 min with the addition of 1000 ng/mL, 500 ng/mL, and 100 ng/mL NGF in the semen extender compared with the control and extender supplemented with 10 ng/mL NGF (p < 0.05).

After 60 min, the addition of 1000 ng/mL and 500 ng/mL Cam-β-NGF resulted in sustained high levels of mass motility, compared to other dosages and the control (p < 0.05) (Fig. 6A). Moreover, viability remained significantly higher after 60 min of incubation with 1000, 500, and 100 ng/mL, in contrast to the control extender and 10 ng/mL supplementation (p < 0.05) (Fig. 6B). Similarly, membrane integrity was notably maintained with supplementation of 1000 ng/mL, 500 ng/mL, and 100 ng/mL of Cam-β-NGF in the semen extender compared to the control and the lower dose (p < 0.05), leading to a higher proportion of healthy spermatozoa at T60 compared to the control and 10 ng/mL supplemented extender (p < 0.05) (Fig. 6C).

4. DISCUSSION

After conservation, spermatozoa lose their traits. In this study, mass motility significantly decreased after 24 hours of storage at 4 °C, and preservation processes affected sperm viability and membrane integrity. This may be due to the elevated levels of unsaturated fatty acids in the sperm plasma membrane and the limited ability of spermatozoa, which contain very little cytoplasm, to fight free radicals. As a result, sperm are highly sensitive to oxidative damage [20, 21].

The plasma membrane of spermatozoa is the primary site affected by low-temperature alterations [22]. Changes in its structure and/or function are often linked to reactive oxygen species (ROS) [23]. Increased ROS production, coupled with a decrease in antioxidants, likely leads to oxidative stress [24], which damages sperm quality and has deleterious effects on spermatozoa. This oxidative damage causes lipid peroxidation, which reduces membrane stability and negatively affects sperm function. Consequently, oxidative stress may explain the decrease in mass motility, viability, and membrane integrity observed in our results. Similar findings have been reported in rats' semen [25-27], ram [28-30], humans [23], alpaca [31-33], llama [34, 35], and camels [36, 37].

In this study, after 24 hours of storage at 4 °C, spermatozoa were treated and subsequently incubated at 36 °C with purified endogenous camel nerve growth factor (NGF) at different concentrations. The goal was to assess whether this protein, which belongs to the neurotrophin family, could help maintain or improve the viability, mass motility, and membrane integrity of camel spermatozoa after storage. The Cam-β-NGF concentrations used in this study (10, 100, 500, and 1000 ng/mL) were selected on the basis of earlier work in camelids, particularly Sari et al. (2021) [34], which reported biological activity of β-NGF within a comparable range.

Cam-β-NGF has been shown to stimulate dromedary camel sperm mass motility in a time- and dose-dependent manner. It is hypothesized that this protein may have receptors on dromedary sperm, potentially enhancing mass motility. These effects are influenced by the presence and distribution of tropomyosin receptor kinase A (TrkA) receptors, which have been identified in the sperm of many species. However, their presence in dromedary camel sperm has not yet been confirmed.

Similar effects have been observed in cooled llama sperm, where supplementation with 10 and 100 ng/mL of exogenous β-NGF improved motility, regardless of incubation duration [34]. In Ilamas, the TrkA receptors were found in the middle piece of the spermatozoon [38], which suggests that β-NGF directly affects sperm motility enhancement in this species. Furthermore, in bulls, these receptors have been identified in the acrosomal cap, nucleus, and tail regions [9]. In addition, Lin et al. [39] reported that NGF could improve the motility of human sperm in vitro by promoting displacement and increasing the proportion of A-grade spermatozoa. Based on these findings in other species, similar mechanisms may occur in dromedary spermatozoa. Immunolocalization studies of Cam-β-NGF receptors (TrkA and/or p75NTR), using techniques, such as immunofluorescence and immunohistochemistry, could provide interesting insights. This research could help develop fertility-enhancing applications in camels.

The addition of Cam-β-NGF maintained sperm viability across all tested doses and the control group at T0 and T30, without significant differences observed. However, after 60 minutes of incubation, sperm viability significantly decreased in the control group and at the 10 ng/mL dose. In contrast, viability remained stable at higher doses (500 and 1000 ng/mL). This result suggests that higher concentrations of Cam-β-NGF may have a protective effect, possibly through its interaction with TrkA receptors. NGF is known to modulate pro-survival factors, preventing cells from entering apoptotic pathways through the activation of the phosphatidylinositide 3-kinase (PI3K) pathway, which plays a crucial role in maintaining cell viability [5]. This pathway is likely involved in sperm survival by inhibiting apoptosis, improving mitochondrial function, and preserving membrane integrity. Sari et al. [34] also reported a positive effect of adding exogenous NGF to cooled llama sperm incubated for 60 minutes with 10 ng/mL. They noted that NGF receptors on sperm are localized in the midpiece, a region directly linked to mitochondrial function [34].

Furthermore, NGF binding to TrkA receptors activates the PI3K pathway, which in turn enhances cell survival [9]. In bulls, Li et al. [9] revealed the presence of NGF and TrkA receptors in ejaculated bovine sperm, and reported that NGF addition (20 ng/L) improved sperm viability after 2 hours of incubation without affecting cellular calcium and acrosome reaction. Although NGF primarily promotes sperm survival through the TrkA receptors, it can also bind to the p75NTR receptor, which may influence apoptosis and necrosis [5]. The balance between these receptor pathways could determine sperm longevity and functionality in the reproductive tract. Further research, including studies with larger sample sizes to overcome the limitations inherent to semen collection in dromedary camels, is needed to elucidate whether the PI3K pathway is directly involved in the NGF-mediated improvements of dromedary sperm viability and to clarify the specific roles of TrkA and p75NTR receptors in sperm survival mechanisms.

Adding Cam-β-NGF improved the sperm membrane integrity of dromedaries in a dose- and time-dependent manner. When sperm are exposed to low temperatures (4 °C), the lipid composition of their membranes can be altered, making them more susceptible to damage and structural instability [40]. This damage affects the membrane’s flexibility and ability to function, which are both important for the sperm’s ability to fertilize an egg. When sperm are stored at low temperatures, it can cause oxidative stress, which overwhelms the sperm’s natural defenses and causes further membrane damage [41]. Interestingly, NGF has been found to help protect the sperm membrane. For example, after thawing bull sperm, those with more NGF mRNA tend to have better membrane quality [42]. This suggests that NGF might reduce the harmful effects of freezing and thawing. Cam-β-NGF could help stabilize the sperm membrane by influencing lipid metabolism and supporting membrane remodeling, which are important for keeping it strong under stressful conditions. In the present experiments, a near-plateau response was detected for most sperm parameters— motility, viability, and membrane integrity— both between 30 and 60 minutes of incubation and between 500 and 1000 ng/mL. To define the full dose- and time-response profile, additional studies with wider concentration intervals and extended incubation periods are required, so that the use of Cam-β-NGF in semen-preservation protocols can be optimized.

CONCLUSION

Our findings suggest that Cam-β-NGF potentially preserves dromedary camel sperm mass motility and viability, and maintains membrane integrity in a time- and dose-dependent manner, likely through a direct interaction with specific receptors on sperm cells. High doses (500 and 1000 ng/mL) showed nearly identical responses, suggesting that testing a broader dose range could pinpoint the true optimum and reveal whether effects decline at higher or lower concentrations. The addition of purified endogenous camel NGF offers a promising approach for the successful short-term preservation of dromedary camel sperm and assisted reproduction techniques. More research is needed to understand exactly how Cam-β-NGF protects sperm and to optimize its use in semen preservation, especially for freezing. Future studies should also examine the effects of Cam-β-NGF on testicular, epididymal, and accessory-gland cells to clarify its role in male-gamete maturation along the entire reproductive tract. Techniques, such as Western blotting or PCR, could help identify receptors and provide further information about their roles.

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