Adenosine supplementation enhances chilled semen quality in Assaf rams: dose-dependent effects on sperm motility, membrane integrity, and morphology

Abstract

Ram semen is sensitive to thermal and oxidative stress due to greater concentrations of polyunsaturated fatty acids (PUFA) in the sperm cell membrane, rendering it inherently fragile during preservation. Adenosine (9-β-D-Ribofuranosyladenine) serves as an essential component in adenosine monophosphate (AMP) and adenosine triphosphate (ATP) synthesis, and plays a key role in sperm energy metabolism and function. This study investigated the effects of adding different adenosine concentrations (0%, 0.5%, 0.75%, and 1%) to Tris-egg yolk extender on the quality of chilled semen of Assaf rams. Semen was collected from seven rams every five days over a one-month period at 7:30 AM, pooled, and extended in Tris-egg yolk, after which adenosine stock solution was added at the designated concentrations. Samples were then cooled to 4°C and stored for 72 h. Motility, viability, membrane integrity, and morphology were assessed at 0, 24, 48, and 72 h. The data were subjected to analysis of variance and comparison of means using Bonferroni test. Incorporating 0.75% adenosine significantly improved semen quality at 48 and 72 h versus other groups (P < 0.05 for all parameters). At 48 and 72 h, respectively, the 0.75% group showed higher total motility (70.9% vs. 60.1%; 56.7% vs. 44.4%), progressive motility (45.2% vs. 34.8%; 39.4% vs. 25.8%), membrane integrity (HOST-positive: 70.3% vs. 64.2%; 68.2% vs. 55.3%), and viability (68.5% vs. 61.2%; 61.8% vs. 52.1%) than the control, with fewer abnormal spermatozoa (12.7% vs. 18.4%; 14.8% vs. 23.5%). Storage time also significantly affected all parameters (P < 0.05), with no significant adenosine × time interaction (P > 0.05). These findings have practical implications for improving sheep reproductive efficiency, given the physiological fragility of the PUFA-rich ovine sperm membrane. Supplementing Tris-egg yolk with 0.75% adenosine is recommended to extend the usable storage life of chilled ram semen up to 72 h, offering greater flexibility in the scheduling and logistics of artificial insemination programs, pending further in vivo fertility trials and comparisons with commercial extenders. Whether this beneficial effect extends to ram semen cryopreservation remains an open question for future research.

This study demonstrates that adenosine supplementation can effectively maintain ram semen quality under chilled conditions, supporting practical applications in sheep breeding. The findings pave the way for potential future advances in semen cryopreservation and assisted reproductive technology.

Introduction

The use of artificial insemination (AI) in sheep breeding has become increasingly common. AI is a valuable reproductive technology; accelerates genetic improvement (De et al. 2015), supports the efforts to control diseases, and enhances reproductive efficiency (Gibbons et al. 2019). This in turn contributes to the achievement of multiple Sustainable Development Goals (SDGs) of the United Nations, particularly SDG 2 (Zero Hunger), SDG 3 (Good Health and Well-being) and SDG 9 (Industry, Innovation, and Infrastructure). However, one of the major challenges to AI programs is the effective preservation of semen quality during storage and transportation (Hameed et al. 2024).

In Palestine, the Assaf sheep breed is one of the most prevalent and economically important sheep breeds, accounting for approximately 36.8% of the national sheep population (PCBS 2021). As a highly productive dual-purpose breed for both milk and meat production, it plays a key role in the Palestinian sheep production sector (Ahmed and Abdallah 2013). Therefore, improving reproductive efficiency and developing effective semen preservation strategies in Assaf rams are important for supporting genetic improvement and sustainable sheep production.

Ram spermatozoa are vulnerable to oxidative stress and cold shock during handling and storage, which causes irreversible damage to the plasma membrane resulting in loss of viability, reduced motility and compromised fertilizing ability (Maxwell and Watson 1996). This susceptibility is mainly associated with the unique lipid composition of ram sperm membranes, which are rich in polyunsaturated fatty acids (PUFAs). Although PUFAs are essential for maintaining membrane fluidity and sperm function, their high susceptibility to lipid peroxidation increases the risk of oxidative damage during storage (Ayala et al. 2014; Carro et al. 2022). To mitigate these negative effects, numerous semen extenders have been developed over the years to provide nutrients and create a protective environment during storage (Bustani and Baiee 2021; Hameed et al. 2024).

Previous studies have demonstrated that acceptable pregnancy rates (30% to 50%) can be achieved using chilled ram semen for AI (Menchaca et al. 2005). Although these results are considered satisfactory compared to frozen-thawed semen (Alvarez et al. 2019), recent studies have focused on the integration of innovative synthetic and natural additives into semen extenders including antioxidants, vitamins, fatty acids, and various bioactive compounds (Allai et al. 2018; Hussain et al. 2018; Bustani and Baiee 2021; Halawa et al. 2025; Khnissi et al. 2025). These efforts aim mainly to improve sperm quality during storage, thereby enhancing fertilization rates and overall reproductive efficiency.

Adenosine (9-β-D-Ribofuranosyladenine) is a purine nucleoside composed of adenine attached to a ribose sugar (Layland et al. 2014). In mammalian cells, adenosine interacts with four subtype receptors belonging to the G-protein coupled-receptors family: A1, A2A, A2B, and A3 (Sheth et al. 2014). Adenosine is known to regulate energy transmission via its interactions with adenosine diphosphate (ADP) and adenosine triphosphate (ATP) (Layland et al. 2014). It plays an energetic role in the regulation of cellular homeostasis and modulation of the release of neurotransmitters (Cunha 2001). Furthermore, adenosine acts as a catalyst to various antioxidants, via activation of A3 receptor (Maggirwar et al. 1994). It also plays a role in a wide range of cell tissues; it modulates the mechanism of T-cell proliferation, and cytokines secretion in immune cells (Sheth et al. 2014).

Previous research have investigated the role of adenosine and its receptors in reproductive physiology. Adenosine plays a vital regulatory role in spermatogenesis, sperm maturation and capacitation as well as acrosome reaction (Bellezza and Minelli 2017). Recently, it was proven that adenosine participates in spermatozoa functions via interaction with A2A receptors in the sperm flagellum (Chen et al. 2024; Zhang and Ma 2024). This interaction regulates the calcium influx, and stimulates the enzyme adenosine cyclase, which subsequently leads to increased intracellular cyclic adenosine monophosphate (cAMP) production and modulates the activity of Adenosine triphosphatases (ATPases) (Fraser and Duncan 1993; Zhang and Ma 2024). This increase in cAMP enhances sperm motility (Romac et al. 1994; Vahidi et al. 2011; Balbach et al. 2018; Chen et al. 2024), and fertilizing ability (Fraser 1981, 1990; Chen et al. 2024). Studies in humans, mice, and other species evaluated the effects of cAMP and ATP as additives on semen quality and showed significantly higher total and progressive motility and better viability (Edwards et al. 2007; Rodríguez-Miranda et al. 2008; Vasudevan and Sztein 2011; Jannatifar et al. 2020).

Beyond its established physiological role in regulating sperm function, adenosine has attracted increasing attention as a potential bioactive molecule for improving semen preservation in several mammalian species. In bovine sperm, adenosine has been evaluated as a capacitation inducer following cryopreservation (Breininger et al. 2010), whereas studies in human sperm have demonstrated that adenosine and its analogues enhance sperm motility and functional activity (Romac et al. 1994).

However, studies incorporating adenosine with semen extenders are still limited. A previous study has evaluated adenosine supplementation at concentrations ranging from 0.5 to 1% and reported potential beneficial effects on sperm quality parameters (Bezerra et al. 2019). Nevertheless, further research is needed to investigate the efficacy of adenosine incorporation under different extender systems, breeds, and storage conditions in ram semen. Therefore, the aim of this study was to investigate the impact of adding different concentrations (0, 0.50, 0.75, and 1%) of adenosine to Tris-egg yolk extender on quality parameters of chilled ram semen at different storage times (0 h, 24 h, 48 h, and 72 h).

Material and methods

The experiment was conducted at Beit-Qad Agricultural Research Station of the Palestinian National Agricultural Research Center (NARC) in the North of the West Bank, Palestine (32.47104506213847, 35.35696929549924) from March to May, 2024. Currently, there are no local regulations governing the animal research and experimentation in Palestine, nor a centralized institutional ethics review board. Therefore, the study was approved by official correspondence from the National Agricultural Research Center (NARC), Ministry of Agriculture, Palestine, and was conducted in accordance with the principles outlined by the NRC (2011) for the care and use of laboratory animals. In the absence of a formal national regulatory framework, the study was additionally designed and reported with reference to internationally recognized standards of animal welfare and reporting transparency, following the general principles of the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments), including random selection of study animals, randomized allocation of pooled semen aliquots across treatment groups, blinding of laboratory personnel to treatment identity during semen quality assessment, and detailed reporting of housing, feeding, and handling conditions. All procedures were performed by trained personnel with the aim of minimizing animal stress and discomfort throughout the study.

Animals

Seven healthy Assaf rams aged 3 to 4 years, weighing 90–110 kg, with body condition score of 3 to 3.5 were randomly selected for the study among rams available for breeding in the research station. Assaf ewes (2–3 years old, 55–60 kg) served as teasers for training rams on the artificial vagina and collection of semen.

Prior to the onset of semen collection, all rams underwent an adaptation period of two weeks to acclimatize them to the collection routine and handling procedures. During this period, rams were progressively trained to mount the artificial vagina in the presence of oestrus-induced Assaf ewe teasers over four training sessions before the start of actual semen collection for the experiment.

Throughout the study, rams were housed individually in pens measuring 2 × 1.5 m, under natural photoperiod conditions, with day length around 13 hours from the onset of experimental semen collection, and ambient temperature averaging approximately 23°C. Animals were fed wheat straw as roughage, supplemented with a commercial concentrate feed containing 14% crude protein, 3.5% crude fat, 12% crude fiber, 1% calcium, 0.2% phosphorus, and 2900 kcal/kg metabolizable energy, along with a vitamin and mineral premix. Feed quantities were formulated according to the nutrient requirements of the National Research Council (NRC 2007) for breeding rams, with free access to fresh water. All rams were subjected to a routine health examination, including body condition scoring and general clinical assessment, prior to inclusion in the study, and were free of any clinical signs of disease or reproductive abnormalities.

Semen collection and initial evaluation

Semen was collected every five days at 7:30 AM for one month (seven collection sessions). Immediately after collection, each ejaculate was evaluated for volume, color, appearance, and pH (pH meter, HAM238100, Hamilton, USA). Then the concentration was checked using a spectrophotometer (Photometer SDM1, Minitube, Germany). Sperm motility was subjectively evaluated through direct observation under a phase-contrast microscope with a magnification 400 × with a warm stage maintained at 37°C (Motic ba310e, Motic group, China). Finaly, morphological evaluation was conducted by adding 10 µL of a fresh semen sample to microtubes containing 100 µL of buffered formal saline. Subsequently, 5 µL of this mixture was placed onto a microscope slide and covered with a glass coverslip, the drop was allowed to spread smoothly for one minute, and at least 200 spermatozoa were counted at a magnification of 1000 × using a phase contrast microscope (Motic ba310e) (Shukla 2011). Ejaculates with a volume ≥ 0.5 ml, normal color and apperance, pH ≤ 6.9, sperm concentration ≥ 2.5 × 10⁹ sperm/ml, sperm motility ≥ 75%, and <10% morphological abnormalities were accepted. Each ram had an acceptable ejaculate each collection day (total of 42 ejaculates over the course of the study). On each collection session, ejaculates from all rams were pooled (to avoid individual variations) in 20 ml sterile plastic culture tubes and evaluated before dilution (Table 1).

Table 1.

Initial quality characteristics of raw pooled ram semen collected over seven collection sessions.

Parameter	Mean ± SD
Volume, mL	8.69 ± 0.29
pH	6.73 ± 0.11
Sperm concentration, × 109 sperm/mL	3.60 ± 0.79
Total motility, %	86.10 ± 3.08
Progressive motility, %	75.79 ± 1.35
Morphological abnormalities, %	5.92 ± 1.09

Preparation of the semen extender

Tris–citric acid–fructose–egg yolk extender was prepared by a laboratory technician, following a two-step protocol (Table 2). First, a Tris buffer solution was formulated by dissolving 3.028 g of Tris (hydroxy-methyl) aminomethane (Sigma Chemical Co., St. Louis, USA), 1.70 g of citric acid monohydrate (Sigma-Aldrich, St. Louis, MO, USA), and 1.25 g of D-fructose (Merck, Darmstadt, Germany) in 100 ml of sterile water (B. Braun Melsungen AG, 2024). The second step involved the preparation of the final extender by combining 80% of the Tris buffer with 20% (v/v) fresh egg yolk, 800 IU/ml of penicillin G and 0.1 mg/ml of streptomycin sulfate (Merck, Darmstadt, Germany) (Rather et al. 2016).

Table 2.

Composition of the Tris–egg yolk extender.

Component	Concentration	Stage
Tris (hydroxymethyl) aminomethane, g/100 mL	3.028	Tris buffer
Citric acid monohydrate, g/100 mL	1.70	Tris buffer
D-Fructose, g/100 mL	1.25	Tris buffer
Sterile distilled water, mL	Up to 100	Tris buffer
Fresh egg yolk, v/v	20%	Final extender
Tris buffer, v/v	80%	Final extender
Penicillin G, IU/mL	800	Final extender
Streptomycin sulfate, mg/mL	0.1	Final extender

Semen dilution

The pooled semen sample from each session (see Semen collection and initial evaluation) was divided into four equal aliquots in plastic vials and diluted 1:4 (semen: extender) gradually and slowly with Tris-egg yolk extender (Nalley and Arifiantini 2011; Bezerra et al. 2019). Adenosine stock solution was then added to each aliquot at the assigned concentration (0%, 0.5%, 0.75%, or 1% v/v), and the four concentrations were randomly assigned to the diluted semen aliquots. After that, each mixture was labled and gently mixed to ensure uniform distribution, then slowly cooled and stored at 4°C in a cooling castle for up to 72 h.

Semen evaluation

Extended semen samples were examined for sperm motility, viability, functional integrity of the spermatozoal membrane and morphology at different storage times (0 h, 24 h, 48 h, and 72 h).

Sperm motility

Sperm motility and Kinematics (total motility, TM; progressive motility, PM; fast progressive motility, FPM; average path velocity, VAP) were evaluated under a phase contrast microscope (Motic ba310e) with customized slides (Lija, IMV Group, Netherland) by Computer-Assisted Sperm Analysis (CASA) using AndroVision software (Fig. 1).

Figure 1.

Representative computer-assisted sperm analysis (CASA) sample report generated by AndroVision software.

Sperm viability

Sperm viability measured as live sperm percentage (LIVE%) was estimated using Eosin-Nigrosine staining technique following Hancock procedure (Shukla 2011). Briefly, 30 g of nigrosine (Nigrosine water soluble, Sigma-Aldrich, Germany) was dissolved in 300 ml of sterilized water (B Braun) by heating and stirring over a digital hotplate stirrer (YR02937, Kalstein, France). Then, 5 g of eosin (Eosin Y disodium salt, Sigma-Aldrich, Germany) was dissolved in the prepared nigrosine solution by the same method. Finally, stain mixture was filtrated and stored at 4°C in a glass vacuum bottle. Sperm smear was prepared by mixing a drop of semen sample with two drops of stain on the end of a warm slide. Then, the mixture was spread immediately with the aid of a second slide. The smear was dried by keeping it on a laboratory heating plate (warming plate with integrated control unit, Minitube, Germany) at 36.8°C (Shukla 2011). A total of 200 spermatozoa were examined under a light microscope (Bx53, Olympus, Japan) using 100 × oil immersion objective lens. As shown in Fig. 2, sperms that were partially or completely stained with purple were considered non-viable (dead); only sperms showing strict exclusion of stain were considered viable (live) (Shukla 2011).

Figure 2.

Representative photomicrograph of live and dead ram spermatozoa (eosin-nigrosin stain).

Plasma membrane functional integrity

Briefly, 20 µl of semen was diluted in 350 µl sodium sulphate–fructose solution (75 mOsm) and incubated in 41°C water bath (Minitube) for 45 minutes, then fixed with 50 µl formalin-buffered saline. Immediately, 10 µl of the prepared sample was used for evaluation (Bacinoglu et al. 2008). A minimum of 200 sperms were counted during evaluation under a phase-contrast microscope (Motic ba310e) at 400 × magnification. As shown in Fig. 3, sperms were categorized based on their response to the hypo-osmotic swelling test (HOST) into two distinct groups: reacted sperm, characterized by a visibly coiled tail, indicating an intact and functional plasma membrane (HOST positive), and non-reacted sperm, which displayed no visible tail coiling (HOST negative), suggesting compromised membrane integrity (Drevius and Eriksson 1966; Ramu and Jeyendran 2013). The percentage of sperms with intact plasma membrane was calculated as the percentage of HOST positive sperms using the following formula:

Figure 3.

Photomicrograph of ram spermatozoa in hypo-osmotic solution showing one HOST-positive sperm with a coiled tail (green circle) and one HOST-negative sperm with a straight tail (red circle).

$$\mathrm{\%}\mathrm{HOST}\mathrm{ }\mathrm{postive}=\left(\frac{\mathrm{Number}\mathrm{ }\mathrm{of}\mathrm{ }\mathrm{reacted}\mathrm{ }\mathrm{sperms}\mathrm{ }}{\mathrm{Total}\mathrm{ }\mathrm{sperms}\mathrm{ }\mathrm{counted}\mathrm{ }}\right)\times 100$$

Morphology evaluation

Morphological assessment was performed using the same eosin-nigrosin stained smears prepared for sperm viability evaluation. The semen sample was examined under a phase-contrast microscope (Motic ba310e) at 1000 × magnification. A minimum of 200 sperms were counted during the observation. As shown in Fig. 4, spermatozoa were classified as having normal or abnormal morphology (Özturkler et al. 2001).

Figure 4.

Photomicrograph of eosin-nigrosin stained ram spermatozoa showing one normal sperm (oval head, green circle) and one morphologically abnormal sperm with a bifid (double) head defect (red circle).

Experimental design

As shown in Fig. 5, the study followed a 4 × 4 factorial treatment structure, combining four adenosine concentrations (0%, 0.5%, 0.75%, and 1% v/v) with four storage/evaluation times (0, 24, 48, and 72 h). Treatments were arranged within a randomized complete block design (RCBD), in which each of the seven semen collection sessions, conducted at 5-day intervals over one month, served as a block. The four adenosine concentrations were randomly assigned to the four aliquots within each session to avoid systematic bias related to aliquot order or position.

Figure 5.

Schematic overview of the experimental design for evaluating the effect of adenosine supplementation on chilled ram semen quality over storage time.

Each of the four diluted aliquots (treatments) was sampled and evaluated at four storage times (0, 24, 48, and 72 h), giving 16 adenosine × time combinations per session. Within each session, every combination was evaluated in two replicates, yielding 32 observations per session and a total of 224 observations across the seven sessions (7 × 4 × 4 × 2).

Statistical analysis

The data were analyzed using SPSS software, version 25.0. Shapiro-Wilk test of normality showed that the distribution was normal for all response variables. Analysis of Variance was carried out using the Mixed procedure based on the following statistical model:

$${\text{Y}}_{\text{ijk}}=\mu +{\text{B}}_{\text{k}}+{\alpha }_{\text{i}}+{\beta }_{\text{j}}+{(\alpha \times \beta )}_{\text{ij}}+{\text{e}}_{\text{ijk}}$$ (1)

where Y_ijk is the response variable, μ is the overall mean, B_k is the random effect of the k^th block (collection session), α_i is the fixed effect of i^th adenosine level, β_j is the fixed effect of the j^th storage time; (α × β)_ij is the interaction effect, and e_ijk is the random error. Pairwise comparisons were performed using the Bonferroni test with significance declared at P < 0.05.

Results

Sperm motility parameters

Table 3 presents the effects of adenosine concentration and storage time on sperm motility parameters. Total motility exhibited a gradual decrease over time across all treatment groups (P < 0.001). In the control group (0% adenosine), total motility dropped significantly from (83.2%) at 0 h to (44.4%) after 72 h, showing the most pronounced decline across all groups. Supplementing Tris -egg yolk extender with 0.75% adenosine significantly alleviated the extent of the decline and maintained the highest TM values (78.9% at 24 h, 70.9% at 48 h, and 56.7% at 72 h; P = 0.021) (Fig. 6a). Both 0.5% and 1% adenosine groups also mitigated the reduction in motility compared with 0% group, although their protective effect was less substantial than that of the 0.75% adenosine.

Table 3.

Effect of incorporating different adenosine concentrations with Tris-egg yolk extender on motility parameters of chilled semen of Assaf rams at different storage times.

Parameters	Adenosine level	Time (h)				P-value
		0	24	48	72	Adenosine	Time	Adenosine * Time
TM, %	0.0%	83.2 ± 3.15a,1	72.3 ± 1.31c	60.1 ± 2.10d	44.4 ± 3.42c	0.021	<0.001	0.059
	0.5%	84.4 ± 1.82a	75.6 ± 1.44b	67.4 ± 1.95b	49.3 ± 2.74b
	0.75%	82.9 ± 3.86a	78.9 ± 1.27a	70.9 ± 2.13a	56.7 ± 3.18a
	1%	83.7 ± 1.29a	76.1 ± 1.28b	64.3 ± 1.86c	48.2 ± 3.16b
PM, %	0.0%	60.3 ± 1.72a	46.7 ± 1.92d	34.8 ± 2.52d	25.8 ± 2.94d	0.032	<0.001	0.054
	0.5%	58.9 ± 2.35a	50.3 ± 2.26b	40.9 ± 2.69b	35.7 ± 2.85b
	0.75%	59.6 ± 2.54a	55.4 ± 1.53a	45.2 ± 2.37a	39.4 ± 3.10a
	1%	57.4 ± 1.16a	47.8 ± 3.14c	37.5 ± 2.55c	33.8 ± 2.77c
FPM, %	0.0%	44.8 ± 1.34a	32.2 ± 3.51c	24.1 ± 2.93c	16.3 ± 2.83d	0.019	<0.001	0.051
	0.5%	43.97 ± 1.15a	34.76 ± 2.93b	27.6 ± 2.48b	24.1 ± 2.51b
	0.75%	43.52 ± 1.82a	39.87 ± 3.92a	34.7 ± 2.81a	28.5 ± 2.74a
	1%	42.31 ± 1.07a	34.12 ± 2.44b	28.1 ± 2.62b	20.4 ± 2.25c
VAP, µm/s	0.0%	87.92 ± 2.49a	61.75 ± 2.20d	48.7 ± 1.73d	31.9 ± 1.74d	0.023	<0.001	0.065
	0.5%	90.15 ± 0.86a	67.13 ± 1.13b	58.3 ± 2.15b	46.2 ± 2.21b
	0.75%	86.87 ± 2.73a	70.41 ± 1.87a	63.8 ± 2.22a	55.9 ± 1.92a
	1%	89.33 ± 1.26a	64.82 ± 1.54c	52.1 ± 1.26c	40.5 ± 2.33c

¹Mean ± SD.

^abcdDifferent superscripts within a column indicate significant differences (p < 0.05) among adenosine levels within each storage time.

Figure 6.

Effect of different adenosine concentrations on sperm motility parameters of chilled Assaf ram semen during storage time (0, 24, 48, and 72 h). (a) Total motility, TM. (b) Progressive motility, PM. (c) Fast progressive motility, FPM. (d) Average path velocity, VAP.

Progressive motility (PM) showed a similar pattern, declining significantly with storage time (P < 0.001). In the 0% adenosine group, PM dropped from 60.3% at 0 h to 25.8% at 72 h, while the 0.75% adenosine maintained significantly higher PM values (P = 0.032) at 72 h (39.4%) (Fig. 6b). Furthermore, both 0.5% and 1% adenosine groups showed intermediate results. Likewise, FPM also declined (P < 0.001) during storage in all groups, but was best preserved in the 0.75% adenosine group (28.5% at 72 h), compared to only 16.3% in the 0% adenosine group (P = 0.019) (Fig. 6c).

Average path velocity (VAP) was also significantly affected by time (P < 0.001) and addition of adenosine (P = 0.023). It decreased over time in all groups, yet the 0.75% adenosine group consistently maintained higher levels (70.4 µm/s at 24 h and 55.9 µm/s at 72 h) compared with the 0% adenosine and other concentrations (P = 0.023) (Fig. 6d).

Interaction effects between adenosine and storage time were not significant (P values ranged from 0.051 for FPM to 0.065 for VAP).

Membrane integrity, viability and morphology

Both storage time and adenosine supplementation showed significant effects (P < 0.05) on sperm membrane integrity, viability, and morphology (Table 4). For HOST positive %, storage time caused a progressive decline in all groups (P = 0.015), and this decline was significantly affected by adenosine concentration (P = 0.033) (Fig. 7a). The 0% adenosine group showed the sharpest decline from 80.1% at 0 h to 55.3% at 72 h, while the 0.75% group maintained the highest levels throughout storage (76.4% at 24 h, and 68.2% at 72 h). However, incorporation of 0.5% and 1% adenosine provided moderate protection (61.6% and 60.8%, respectively, at 72 h).

Table 4.

Effect of incorporating different adenosine concentrations with Tris-egg yolk extender on sperm membrane integrity, viability, and morphology of chilled semen of Assaf rams at different storage times.

Parameters	Adenosine level	Time (h)				P-value
		0	24	48	72	Adenosine	Time	Adenosine * Time
HOST positive, %	0.0%	80.1 ± 5.15a,1	72.4 ± 4.35b	64.2 ± 3.15 cd	55.3 ± 4.42c	0.033	0.015	0.064
	0.5%	79.3 ± 4.89a	72.2 ± 4.12b	66.5 ± 4.05b	61.6 ± 4.36b
	0.75%	79.0 ± 4.76a	76.4 ± 3.78a	70.3 ± 4.13a	68.2 ± 3.85a
	1%	79.9 ± 5.09a	72.9 ± 3.98b	65.4 ± 3.26bc	60.8 ± 3.95b
LIVE, %	0.0%	78.4 ± 1.22a	69.5 ± 2.15d	61.2 ± 1.42d	52.1 ± 1.73d	0.025	0.011	0.057
	0.5%	77.4 ± 1.71a	70.3 ± 2.11b	65.8 ± 1.89b	58.9 ± 1.85b
	0.75%	76.6 ± 1.34a	73.6 ± 1.85a	68.5 ± 1.37a	61.8 ± 1.29a
	1%	77.1 ± 1.28a	70.3 ± 2.04b	63.1 ± 1.75c	55.3 ± 1.72c
Abnormal morphology, %	0.0%	8.1 ± 1.14a	14.7 ± 1.21a	18.4 ± 1.33a	23.5 ± 1.74a	0.022	0.017	0.061
	0.5%	7.9 ± 1.35a	12.1 ± 1.13b	15.2 ± 1.18c	17.1 ± 1.51c
	0.75%	7.8 ± 1.10a	10.9 ± 1.29c	12.7 ± 1.51d	14.8 ± 2.74d
	1%	8.3 ± 1.17a	12.5 ± 2.44b	16.4 ± 1.61b	19.6 ± 1.35b

¹Mean ± SD.

^abcdDifferent superscripts within a column indicate significant differences (P < 0.05) among adenosine levels within each storage time.

Figure 7.

Effect of different adenosine concentrations on sperm membrane integrity, viability, and morphology of chilled Assaf ram semen during storage time (0, 24, 48, and 72 h). (a) HOST-positive spermatozoa, membrane integrity. (b) Sperm viability. (c) Abnormal sperm morphology.

Sperm viability (LIVE %) also decreased significantly with storage time (P = 0.011), with adenosine concentration also exerting a significant effect (P = 0.025) (Fig. 7b). The 0% adenosine group dropped from 78.4% to 52.1% by 72 h. The 0.5% adenosine slowed this decline, with viability remaining at 58.9% at 72 h, while 1% adenosine preserved a slightly lower level (55.3%). Recurrently, the 0.75% adenosine group maintained the highest viability (61.8% at 72 h).

As expected, abnormal sperm morphology increased significantly with storage time (P = 0.017), and was significantly influenced by adenosine concentration (P = 0.022) (Fig. 7c). The 0% adenosine group reached the highest abnormality level (23.5%) at 72 h. Adenosine supplementation elicited a dose-dependent attenuation of the rate of increase. At 72 h, abnormalities were 17.1% in the 0.5% adenosine group and 19.6% in the 1% adenosine group, compared with only 14.8% in the 0.75% adenosine group. This indicates that although both 0.5% and 1% provided improvements over the control, the 0.75% adenosine gave the best protection for sperm morphology across all storage times.

No significant interaction effects were found between adenosine level and storage time for membrane integrity (P = 0.064), sperm viability (P = 0.057), and abnormal sperm morphology (0.061).

Discussion

Ovine spermatozoa are among the most cold-shock- and peroxidation-sensitive gametes of the domestic species. Compared with bull, boar, and human sperm, ram sperm membranes combine a low cholesterol-to-phospholipid ratio with high proportion of long-chain PUFA, particularly arachidonic acid (20:4) and docosahexaenoic acid (22:6), which together account for a large share of the unsaturated fatty acid pool of the plasma membrane (Parks and Lynch 1992; Salamon and Maxwell 1995). However, seminal plasma provides ram spermatozoa with a first line of antioxidant defense, albeit at comparatively modest concentrations of enzymes such as superoxide dismutase, glutathione peroxidase and catalase (Abu-Erreish et al. 1978; Mann and Lutwak-Mann 1981). Once semen is diluted for storage, this already-limited protection is further diminished, leaving the arachidonic and docosahexaenoic acid residues in the sperm membrane particularly exposed to attack by reactive oxygen species (ROS). The resulting lipid peroxidation, marked by the accumulation of malondialdehyde and other reactive aldehydes, gradually erodes membrane fluidity, disrupts ion-channel function, and undermines motility (Salamon and Maxwell 2000). It is this inherent, species-specific fragility that makes oxidative protection, whether through classical antioxidants or bioactive additives such as adenosine, so central to preserving ram semen quality during both liquid storage and cryopreservation.

In this study, storage time negatively affected sperm parameters for all groups. Semen quality of rams remained relatively stable during the first 24 hours of storage at 4°C. Many studies have demonstrated that Tris-egg yolk extenders are effective in maintaining semen quality during short-term storage (Salamon and Maxwell 2000; Hameed et al. 2024). However, a progressive decline in semen quality is observed beyond 48 and 72 hrs. This reduction is largely ascribed to oxidative stress, lipid peroxidation and reduced mitochondrial activity, which impair sperm membrane stability and function (Hameed et al. 2024). This time-dependent pattern of deterioration observed in the present study is consistent with findings from previous studies evaluating bioactive additives in semen extenders for small ruminants. For instance, vitamin C, vitamin E, and bee-bread extract supplementation in ram and buck semen stored at 4 to 5°C has been reported to improve sperm quality characteristics and mitigate oxidative stress during storage; however, these protective effects were not sustained over extended periods, as a progressive decline in sperm function became evident after 48 to 72 h (Sharaf et al. 2022; Akhter et al. 2023). Adenosine performs a pivotal role in sperm cell mechanisms and functions. It serves as an essential component in AMP and ATP synthesis (Layland et al. 2014), which in turn affects sperm functions. In the present study, supplementing the Tris-egg yolk extender with 0.75% adenosine improved all key semen parameters at 24, 48, and 72 h of storage at 4°C relative to the control and to the other tested concentrations, with this group conferring the greatest resistance to the physiological deterioration associated with prolonged storage. To our knowledge, this is among the first studies to evaluate adenosine supplementation in a Tris-egg yolk extender, in Assaf ram breed, and over an extended storage period of up to 72 h.

The observed improvement in semen parameters aligns with a growing body of literature on adenosine’s beneficial role in reproductive biology (Edwards et al. 2007; Vahidi et al. 2011; Bellezza and Minelli 2017; Bezerra et al. 2019; Jannatifar et al. 2020). Mechanistically, adenosine interacts with A2A receptors, stimulating cAMP production and ATPase activity within the sperm tail membrane (Fraser 1990; Romac et al. 1994; Liguori et al. 2005; Vahidi et al. 2011; Zhu et al. 2018; Zhang and Ma 2024). Recent work has localized these receptors directly to the sperm tail, showing that their engagement also modulates intracellular calcium handling via the CatSper channel, linking cAMP/PKA signaling to the cell’s fertilizing capacity (Chen et al. 2024). This receptor-mediated mode of action sets adenosine apart from the antioxidant-centred additives more commonly evaluated in ram semen extenders, such as melatonin, astaxanthin, and vitamin E, whose principal mechanism is ROS scavenging rather than signaling (Dai et al. 2019; Sohail et al. 2024). Given that the two classes act largely through non-overlapping pathways, adenosine may prove more valuable as a complement to, rather than a substitute for, conventional antioxidant supplementation. Our findings are in agreement with previous studies, which found that adenosine modulates sperm motility via altering lactate and ATP levels (Hurtado de Llera et al. 2012; Zhu et al. 2018), and parallel similar improvements observed when adenosine was added to the semen of infertile men (Vahidi et al. 2011) and to Andromed-extended Santa Inês ram semen during liquid storage (Bezerra et al. 2019).

Membrane functional integrity, viability, and normal morphology were also significantly improved in adenosine-supplemented samples, an effect likely attributable to adenosine’s multifaceted biological actions: it stabilizes the cellular membrane by limiting lipid peroxidation (Correia and Vale 2024) and regulating lipid homeostasis (Koupenova and Ravid 2013), effects that together help preserve sperm integrity under prolonged storage stress. This protective role is not unique to reproductive cells; the same A2A-receptor/cAMP/PKA axis has been shown to regulate autophagy flux and apoptosis under ischemic stress in other tissues (Xia et al. 2022), lending broader biological support to the improvements in viability and membrane integrity observed here.

The optimal concentration of adenosine was 0.75%, at which the highest sperm motility, viability, normal morphology and membrane integrity were observed at different storage times, whereas 1% adenosine, showed reduced efficacy, demonstrating a partially reversed or counteractive effect compared with 0.75% adenosine. This dose-dependent pattern is consistent with previous studies showing that adenosine, acting as a purinergic signaling molecule, is beneficial at low-to-moderate concentrations but deleterious at higher concentrations, with declines in motility and membrane damage reported at excessive doses (Rodríguez-Miranda et al. 2008; Bellezza and Minelli 2017; Bezerra et al. 2019; Jannatifar et al. 2020).

No significant interaction effects between adenosine level and storage time were found for any of the studied sperm quality parameters. Rather than a limitation of the experimental design, this finding carries biological meaning: it implies that adenosine does not merely buy sperms additional time before an unavoidable deterioration sets in, but instead recalibrates the baseline resilience of the cell to the stresses of storage from the outset. The parallel, non-diverging trajectories observed across concentrations and storage durations suggest that adenosine’s mode of protection is intrinsic to the cell’s physiological state rather than a function of elapsed time, reinforcing its candidacy as a genuine modifier of sperm survival rather than a temporary buffer against decline. This interpretation, however, remains inferential and would benefit from further direct investigation.

Study limitations

While the present findings offer clear evidence of adenosine’s beneficial effects under controlled laboratory conditions, three aspects merit consideration for future work. The evaluation relied on in vitro sperm parameters rather than in vivo fertility outcomes, which would ultimately confirm the practical significance of these improvements under field insemination conditions. The sample size, while adequate to detect the treatment and storage-time effects reported, was drawn from seven rams and would benefit from expansion across breeds and individual fertility profiles in subsequent studies. Finally, adenosine was evaluated against a Tris-egg yolk base extender; comparison with commercial extenders would help position its relative value for producers. These represent natural next steps rather than shortcomings of the present design, and are addressed in the future research directions below.

Future research perspectives

Building on the present liquid-storage findings, a natural next step would be to explore adenosine supplementation within ram semen cryopreservation protocols, where the PUFA-rich ram sperm membrane faces considerably greater cold-shock and peroxidative stress than during 4°C liquid storage. Adenosine’s combined bioenergetic, receptor-signaling, and membrane-stabilizing actions make it a promising candidate for cryoprotectant formulations aimed at improving post-thaw motility and membrane integrity. Complementary avenues worth pursuing include in vivo fertility trials following artificial insemination with adenosine-supplemented semen, studies across larger and more genetically diverse ram cohorts, comparative work against commercial extenders, and mechanistic investigations, such as direct measurement of intracellular cAMP and Ca²⁺ dynamics, to further test the hypotheses proposed above for the effect observed at 1% adenosine.

Conclusions

Semen quality remained relatively stable during the first 24 hours of storage in Tris-egg yolk extender, after which a progressive decline became evident, particularly beyond 48 and 72 hours. Adding adenosine, particularly at 0.75%, exerted a protective effect against this time-dependent deterioration, improving motility, membrane integrity, and viability at 48 and 72 hours relative to the control and to other tested concentrations. Notably, a higher concentration (1%) showed reduced efficacy, indicating that adenosine’s benefit is dose-dependent rather than simply dose-proportional. These findings suggest that adenosine may be a valuable additive for extending the usability of chilled ram semen, though further in vivo studies are needed to confirm its effects on fertility outcomes. The results of this study have significant implications for the AI industry as a means to improving livestock reproductive efficiency, ultimately improving production efficiency and global food security in alignment with the SDGs of the United Nations.

Glossary

List of abbreviations

AI

artificial insemination

SDGs

sustainable development goals

ADP

adenosine diphosphate

ATP

adenosine triphosphate

AMP

adenosine monophosphate

PUFA

polyunsaturated fatty acids

cAMP

cyclic adenosine monophosphate

ATPases

adenosine triphosphatases

NARC

Palestinian National Agricultural Research Center

CASA

computer-assisted sperm analysis

TM

total motility

PM

progressive motility, FPM, fast progressive motility

VAP

average path velocity

LIVE%

live sperm percentage

HOST

hypo-osmotic swelling test

RCBD

randomized complete block design

ANOVA

analysis of variance

Funding

The authors received no specific funding for this work.

Author contributions

Mousa Khalil (Conceptualization, Investigation, Formal analysis, Methodology, Writing—original draft) and Jihad Abdallah (Conceptualization, Methodology, Supervision, Writing—review & editing).

Conflicts of interest

The authors declare no conflict of interest.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.