Enhancing sperm resilience: protective effects of ectoine on post-thaw bovine sperm quality under environmental stress conditions

TL;DR

Ectoine, a natural osmolyte, protects bovine sperm from environmental stress without toxicity, showing promise for reproductive biotechnology.

Contribution

This study is the first to demonstrate ectoine's protective effects on sperm under stress conditions.

Findings

Ectoine at 0.5 and 5 mM maintained sperm motility and viability under heat stress.

Ectoine improved sperm viability in hypoosmotic conditions for bulls with poor osmotic resistance.

Sperm treated with 50 mM ectoine showed higher motility in cervicovaginal mucus.

Abstract

Ectoine is a small, amino acid-derived osmolyte produced by extremophilic bacteria that acts as a compatible solute, protecting cellular macromolecules and structures from extreme environmental stress without disrupting essential cellular functions. The aim of this study was to evaluate the biocompatibility of ectoine with bull sperm and to assess the potential of ectoine to enhance the resilience of sperm under varying stress conditions. Thawed bovine sperm in the presence (0.5, 5, and 50 mM) or absence (control; 0 mM) of ectoine were subjected to a biocompatibility test (37 °C for 6 h; n = 8 bulls), heat stress (39 or 42 °C for 6 h; n = 8 bulls) or osmotic stress (150 or 400 mOsm for 15 min; n = 12 bulls), whereby motility and kinematic parameters, as well as viability, acrosome integrity, and membrane fluidity by flow cytometry were assessed. Sperm motility in cervicovaginal mucus (37 °C for 3 h; n = 6 bulls) was also assessed. All results are reported as mean ± SEM. Ectoine displayed a non-toxic effect across all motility and functional parameters (viability, acrosome integrity, and membrane fluidity). Nonetheless, a reduction in kinematic parameters, including straight line velocity (VSL), average path velocity (VAP) and straightness (STR) was observed at 50 mM ectoine (P < 0.05). Under heat stress at 39 and 42 °C, ectoine concentrations of 0.5 and 5 mM maintained motility and viability, comparable to controls across all time points (P > 0.05). In hypoosmotic conditions (150 mOsm), individual bulls displayed different degrees of osmotic resistance. In those bulls with poor osmotic resistance (n = 4), ectoine (0.5 and 5 mM) maintained sperm viability similar to the 0 mM control (P > 0.05). However, the viability of sperm incubated with 50 mM solute was 2-fold higher relative to the control (P < 0.001). In hyperosmotic conditions, addition of ectoine to sperm prior to exposure did not affect the total motility or viability compared to the no ectoine treatment (P > 0.05). When sperm were incubated in cervicovaginal mucus, there was an effect of ectoine treatment. Sperm treated with 50 mM ectoine exhibited higher motility throughout incubation compared to the control (0 mM) (P < 0.05). In conclusion, these findings establish ectoine as a promising candidate for improving sperm resilience and warrants further studies to assess additional protective effects of ectoine.

Ectoine, a naturally occurring osmolyte, has been shown to protect various cell types under extreme environmental stress, but has never been examined for the protection of sperm. This research showed ectoine’s potential to enhance sperm resilience under stress, highlighting its promise as a protective agent in reproductive biotechnology.

Figures

Representative histograms and scatter plots obtained following the assessment of viability using live/dead dual staining. The areas of the histograms or plots highlighted by colored boxes identify the populations analyzed in this study. The blue-colored box (A) highlights a sperm subpopulation positive for SYTO 16. The highlighted population indicated by the green-colored box (B) identifies the sperm population negative for Propidium Iodide (PI). The bottom right quadrant, colored in red (C) on the scatter plot highlights all sperm that are PI-/SYTO16+ and therefore determined as viable.

Representative histograms and scatter plots illustrating the simultaneous evaluation of (A) viability (B, C) membrane fluidity and (D, E) acrosome integrity using 4′,6-diamidino-2-phenylindole (DAPI), merocyanine 540 (M540), and PNA–Alexa Fluor 647 (PNA-AF-647), respectively. Highlighted regions within the histograms and plots (green boxes) indicate the specific sperm populations analyzed. For membrane fluidity, the green box on the histogram (B) identifies sperm with low membrane fluidity, while the corresponding scatter plot (C) shows the percentage of sperm which are live and have low membrane fluidity. Similarly, for acrosome integrity, the green box on the histogram (D) denotes sperm with an intact acrosome, and the scatter plot (E) shows the percentage of sperm which are live and have an intact acrosome.

Effect of ectoine on bull sperm post-thaw during heat stress. Total motility of (A) sperm incubated at 39 °C and (B) sperm incubated at 42 °C and assessed at 0, 3, and 6 h (n = 8 bulls). abDifferent superscripts indicate a significant difference (P < 0.05). Data are displayed as mean ± SEM. ns = non-significant.

Effect of ectoine on protecting sperm against hypoosmotic conditions. (A) total motility and (B) viability of bull sperm incubated at 37 °C for 15 min in 150 mOsm mTALP following exposure to 0, 0.5, 5, and 50 mM ectoine (n = 12 bulls). Iso media was mTALP media of 300 mOsm osmolarity which acted as the control. Data are displayed as mean ± SEM. abcDifferent superscripts indicate a significant difference within osmotic tolerance groups (P < 0.05). Bulls were assigned to categories of either high, medium or low (n = 4 bulls per category) osmotic tolerance based on the sperm viability at 150 mOsm in the 0 mM ectoine treatment.

Total motility of frozen-thawed bull sperm in the absence or presence of ectoine, incubated for 3 h in oestrus phase cervicovaginal mucus (n = 6 bulls). Media control in mTALP was included. Sperm were assessed subjectively using a phase contrast microscope at a magnification of 200×. abcDifferent superscripts indicate a significant difference (P < 0.05). Data are displayed as mean ± SEM. ns = non-significant.

Tables

Table 1. Range of post-thaw sperm functional variables assessed in bull semen in the presence of ectoine (n = 8 bulls). Samples were assessed at 0, 3, and 6 h post incubation. Results are presented as the mean ± SEM.

Parameter	Time of incubation (h)	Ectoine Concentration (mM)				Effect of treatment	Effect of time	Treatment by time interaction
		0	0.5	5	50	P value	P value	P value
Total motility (%)	0	51.8 ± 3.27	47.4 ± 4.47	51.3 ± 3.73	40.6 ± 3.73	ns	<0.001	ns
	3	33.9 ± 3.94	37.7 ± 5.47	34.5 ± 5.04	32.7 ± 4.65
	6	31.6 ± 3.54	36.1 ± 2.76	36.4 ± 4.51	24.5 ± 2.86
Progressive motility (%)	0	43.0 ± 2.29	39.1 ± 3.20	39.4 ± 2.92	31.2 ± 4.05	ns	<0.001	ns
	3	28.4 ± 3.68	29.8 ± 4.02	28.6 ± 4.02	24.1 ± 3.91
	6	25.1 ± 3.44	29.0 ± 3.47	28.0 ± 3.80	14.7 ± 2.97
Curvilinear velocity (VCL; μm/s)	0	175.6 ± 9.34	169.7 ± 9.11	165.4 ± 11.23	168.3 ± 13.20	ns	<0.001	ns
	3	166.7 ± 7.15	164.4 ± 6.06	166.0 ± 6.31	131.9 ± 3.81
	6	146.8 ± 5.51	145.2 ± 8.58	142.0 ± 8.08	118.9 ± 7.70
Straight-line velocity (VSL; μm/s)	0	100.0 ± 5.57	97.3 ± 5.60	89.7 ± 5.64	80.0 ± 9.89	<0.001	<0.001	<0.01
	3	98.4 ± 4.69	97.2 ± 4.92	99.1 ± 4.19	56.9 ± 2.68
	6	80.5 ± 5.82	83.0 ± 6.32	78.6 ± 6.45	44.3 ± 6.13
Average path velocity (VAP; μm/s)	0	112.3 ± 5.80	109.4 ± 5.57	102.8 ± 6.04	96.2 ± 9.28	<0.001	<0.001	<0.05
	3	108.2 ± 4.53	107.0 ± 4.44	108.3 ± 3.97	71.2 ± 2.16
	6	91.8 ± 5.22	92.5 ± 6.04	88.4 ± 5.83	60.3 ± 5.77
Amplitude of lateral head movement (ALH; μm)	0	7.1 ± 0.52	7.0 ± 0.51	6.9 ± 0.62	8.9 ± 0.45	<0.05	ns	ns
	3	6.9 ± 0.36	6.8 ± 0.30	6.8 ± 0.38	8.0 ± 0.27
	6	7.0 ± 0.30	6.7 ± 0.44	6.7 ± 0.44	7.7 ± 0.29
Beat cross frequency (BCF; Hz)	0	32.6 ± 2.36	32.2 ± 2.40	32.6 ± 2.13	25.6 ± 2.10	<0.05	<0.001	ns
	3	33.2 ± 1.86	32.7 ± 1.58	32.1 ± 1.81	20.3 ± 0.89
	6	28.7 ± 1.96	27.8 ± 1.78	27.3 ± 2.06	20.2 ± 1.04
Linearity (LIN; %)	0	58.1 ± 2.68	58.4 ± 3.15	56.7 ± 3.15	47.9 ± 3.21	<0.001	<0.05	ns
	3	58.1 ± 1.56	59.1 ± 1.65	59.0 ± 1.62	43.8 ± 1.96
	6	53.6 ± 2.67	57.5 ± 3.10	55.2 ± 3.33	37.0 ± 2.67
Straightness (STR; %)	0	87.1 ± 1.19	86.7 ± 1.42	85.5 ± 1.67	80.1 ± 2.33	<0.001	<0.001	<0.001
	3	88.2 ± 0.96	88.3 ± 0.96	89.0 ± 1.03	78.3 ± 2.09
	6	84.1 ± 1.90	87.4 ± 1.87	86.2 ± 2.23	70.6 ± 2.61
Wobble (WOB; %)	0	65.6 ± 2.33	66.1 ± 2.78	64.8 ± 2.65	58.1 ± 2.50	<0.001	<0.05	ns
	3	64.7 ± 1.21	65.7 ± 1.38	65.3 ± 1.20	54.9 ± 1.02
	6	62.1 ± 2.03	64.4 ± 2.34	62.7 ± 2.50	51.3 ± 1.81
Viability (%)	0	53.0 ± 3.81	50.6 ± 4.37	51.1 ± 4.00	54.8 ± 3.67	ns	<0.001	ns
	3	43.6 ± 4.94	43.4 ± 5.38	45.2 ± 5.24	48.2 ± 4.19
	6	40.2 ± 4.52	41.6 ± 4.89	40.4 ± 5.94	37.9 ± 4.00
Acrosome integrity (%)	0	78.9 ± 2.22	76.1 ± 2.79	76.2 ± 2.09	76.3 ± 1.82	ns	<0.001	ns
	3	75.6 ± 0.93	77.1 ± 2.64	79.5 ± 2.74	77.0 ± 1.43
	6	46.8 ± 2.39	48.0 ± 0.88	47.6 ± 1.33	45.0 ± 3.74
Live and low membrane fluidity (%)	0	50.5 ± 6.94	51.0 ± 6.14	51.3 ± 6.95	53.3 ± 6.59	ns	<0.05	ns
	3	47.1 ± 3.44	48.1 ± 3.00	49.6 ± 2.53	44.0 ± 1.77
	6	39.2 ± 2.36	41.8 ± 2.16	43.2 ± 3.00	39.2 ± 3.83

Full text

Introduction

Sperm face a remarkable journey through the female reproductive tract to reach the oocyte. This journey is long and tortuous, with numerous physical, chemical, and biological barriers that sperm must overcome to achieve fertilization. These barriers include navigating the complex anatomy of the female reproductive tract, interaction with its epithelial lining and secretions, swimming against an outward flow of viscous, negatively charged mucus while avoiding immune attack by the adaptive female immune response (Miller 2018). Collectively, these challenges impose significant stress on sperm, inhibiting sperm migration through the female reproductive tract and reducing the number of functional sperm capable of reaching the oocyte for fertilization. Therefore, stress tolerance is crucial to ensure that sperm retain their functional integrity, motility, and fertilization capacity despite exposure to these diverse environmental challenges. As sperm are terminally differentiated, possessing a highly condensed nucleus with limited cytoplasm, and are transcriptionally and translationally inactive (Grunewald et al. 2005), adapting to these conditions is highly challenging.

Interestingly, a class of molecules known as compatible solutes have been extensively studied for the pivotal role they play in protecting macromolecules and somatic cell structures from extreme environmental stress, such as high salinity, extreme temperatures, and osmotic imbalances (Pastor et al. 2010). Among these solutes, ectoines have emerged as one of the most promising, and have been shown to display the most effective stabilizing properties (Lippert and Galinski 1992). Ectoine, along with its related derivative hydroxyectoine, protects cell membranes, proteins, enzymes, and nucleic acids from dehydration through the formation and stabilization of hydro-layers around them via a mechanism known as the “preferential exclusion model” (Zaccai et al. 2016). In this model, ectoine is excluded from the immediate protein hydration shell and membrane hydration layers to the bulk region, where they bind to water molecules, forming extensive stable water-ectoine clusters (Zaccai et al. 2016). These clusters maintain or even increase the hydration of macromolecules, preventing their denaturation (Lippert and Galinski 1992; Knapp et al. 1999), while also enhancing the hydration and fluidity of the cell membranes (Harishchandra et al. 2010; Herzog et al. 2019). Several biophysical studies have demonstrated that ectoine exhibits superior water-binding properties compared to other osmoprotectants, such as glycerol (Graf et al. 2008).

Critically, compatible solutes like ectoine do not interfere with cellular metabolism or physiological processes, making them ideal for therapeutic applications (Galinski 1993). The marked protective effects of ectoine on biomolecules, along with its non-toxic properties have led to the formulation of several over-the-counter medicines, such as nasal, mouth and throat sprays, eye drops, and skin lotions. Moreover, ectoine has been shown to be effective in the treatment of certain disease states, such as preexisting colitis, through the protection and restoration of intestinal barrier integrity (Abdel-Aziz et al. 2013). It has also been reported to protect ileal mucosa and muscularis against ischemia and reperfusion injury (Wei et al. 2009). Despite ectoine’s well-documented protective effects, no published studies have investigated its impact on sperm function or its potential to aid sperm transport through the complex environment of the female reproductive tract. Ectoine’s reported membrane-stabilizing capabilities (Herzog et al. 2019) may assist sperm in navigating the complex female reproductive tract environment, ultimately improving their transport and survival. Given that the interaction between ectoine and sperm cells has not been previously investigated, establishing the biocompatibility of ectoine on sperm is essential for its potential use in assisted reproduction.

Therefore, this study aimed to evaluate the biocompatibility of ectoine with bovine sperm and to explore its potential to enhance sperm resilience under stress-induced conditions. A series of in vitro experiments were designed to further understand the ­response of sperm against stresses, such as heat, osmotic pressure, and prolonged exposure to cervical mucus, with the goal of evaluating ectoine’s ability to improve sperm stress tolerance against these parameters.

Materials and methods

All procedures were performed in vitro using frozen bull semen collected under commercial conditions from an EU-licensed and approved semen processing center. After routine processing, semen straws were donated for research purposes. No research procedures were conducted on live animals, and no procedures deviated from standard commercial semen collection practices.

Chemicals and media

All chemicals were purchased from Sigma Aldrich (Arklow, Co Wicklow, Ireland) unless otherwise stated.

The basal medium used for all experiments was a modified Tyrode’s Albumin Lactate Pyruvate (mTALP) medium supplemented with lactate and pyruvate and devoid of bicarbonate and ­bovine serum albumin (BSA) (Parrish et al. 1989). The mTALP consisted of 2 mM CaCl_2_, 3.1 mM KCl, 0.4 mM MgCl_2_, 125 mM NaCl, 0.3 mM NaH_2_PO_4_, 15 mM HEPES, 21.6 mM sodium lactate, and 1 mM sodium pyruvate. The medium was also supplemented with 0.5 mg/mL polyvinyl alcohol. Before using mTALP in experiments, the pH of the medium was adjusted to 7.3 with 1N NaOH and to an osmolarity of 300 to 320 mOsm following which it was passed through a filter (0.22 μm pore size).

Semen collection, processing and preparation

Semen was collected from mature Holstein Friesian bulls at a commercial stud using an artificial vagina after stimulation and mounting on a teaser bull. The ejaculate was fully extended in BullXcell (IMV Technologies, L’Aigle, France) and packaged into 0.25 mL semen straws (IMV Technologies), at a concentration of 80 × 10^6^ sperm per mL. Straws were filled, sealed, and printed as per routine procedures. Straws were then cooled to 4 °C over 3 h and were frozen to −140 °C as follows: −5 °C per min from +4 °C to −10 °C, −40 °C per min from −10 °C to −100 °C and thereafter −20 °C per min from −100 °C to −140 °C in a programmable freezer (IMV Technologies), followed by submersion and storage in liquid nitrogen at −196 °C until use. Four straws from each ejaculate of each bull were assessed subjectively immediately post-thaw via standard microscopic techniques for total and gross motility (Murphy et al. 2018). Post-thaw quality control thresholds were set at ≥ 50% total motility and a gross motility score of ≥ 3 on a 5-point scale (1 = twitching/no forward progression; 5 = excellent forward progressive motility). Ejaculates that passed the quality control were then donated to the project. Prior to use, semen straws were thawed in a 37 °C water bath for 30 s, then washed free of the egg yolk-based extender by centrifugation at 300 × g for 5 min. Sperm concentration was determined using a hemocytometer, and samples were resuspended in mTALP to achieve the desired concentration.

Study design

Experiment 1: assessment of the effect of ectoine on in vitro sperm functional parameters

The objective of this experiment was to assess the biocompatibility of ectoine (product number 81619, Sigma Aldrich) on sperm function over time. Following washing, frozen-thawed sperm (n = 8 bulls) were diluted to a concentration of 20 × 10^6^ sperm per mL in 200 μL mTALP in the presence of 0 (control), 0.5, 5, and 50 mM ectoine. Sperm were then incubated at 37 °C for 6 h and at 0, 3, and 6 h an aliquot was taken for motility and kinematic parameters assessment using Computer Assisted Sperm Analysis (CASA). Viability, acrosome integrity, and membrane fluidity were assessed using flow cytometry.

Experiment 2: assessment of the protective effects of ectoine on heat-stressed sperm

The objective of this experiment was to assess the protective effect of ectoine on heat-stressed sperm. Following washing, frozen–thawed sperm (n = 8 bulls) were preincubated for 1 h at 37 °C in mTALP medium supplemented with ectoine at 0 (control), 0.5, 5, or 50 mM at a final sperm concentration of 20 × 10^6^ sperm/mL. Sperm were then incubated at 39 and 42 °C for a further 6 h and at 0, 3, and 6 h an aliquot was taken for motility and kinematic parameters assessment using CASA. Viability was assessed using flow cytometry.

Experiment 3: assessment of the protective effects of ectoine against osmotic stress

Experiment 3a: optimization of the solutions used for hypoosmotic and hyperosmotic media

In order to determine sperm susceptibility to osmotic stress, a preliminary experiment was conducted to assess the optimum hypoosmotic or hyperosmotic media to use. A range of hypoosmotic media were prepared by diluting isoosmotic mTALP (300 mOsm/kg) with dH_2_O to produce a range of hypoosmotic media, namely 300, 250, 200, 150, 100, and 50 mOsm. Osmolarities were determined using a KNAUER Semi-Micro Osmometer K-7400 (KNAUER, Berlin, Germany). Following washing, frozen-thawed sperm (n = 6 bulls) were exposed to the range of hypoosmotic media for 15 min (Murphy et al. 2016), following which motility (CASA), viability (flow cytometry), and degree of tail folding (microscopy) were assessed. Tail folding indicates the functional integrity of the sperm’s plasma membrane. Briefly, to assess tail folding, the sperm sample was mixed 1:1 with Nigrosin-Eosin ­following which 10 µL of the mixture was spread on a slide and ­allowed to air dry. A total of 100 sperm were categorized per sample by tail appearance into 1) normal (no tail or tip folding), 2) tip coiling, 3) 50% tail folding, or 4) entire tail folding, assessed according to Holmes et al. (2020). Based on the results, 150 mOsm was selected as the threshold osmolarity for all further investigations (Supplementary Figure S1). Similarly, a range of hyperosmotic media were prepared by diluting isoosmotic mTALP (300 mOsm/kg) with 5M NaCl to produce a range of hyperosmotic media, namely 300, 400, 500, and 600 mOsm. Osmolarities were determined using a KNAUER Semi-Micro Osmometer K-7400. Following washing, frozen-thawed sperm (n = 6 bulls) were exposed to the range of hyperosmotic media for 15 min, following which motility (CASA) and viability (flow cytometry) were assessed. Based on the results, 400 mOsm was selected as the threshold osmolarity for all further investigations (Supplementary Figure S2).

Experiment 3b: effect of ectoine on protecting sperm against ­osmotic stress (low and high osmolarity)

The objective of this experiment was to assess the protective effect of ectoine on sperm under hypoosmotic or hyperosmotic stress. Following washing, frozen-thawed sperm (n = 12 bulls) were preincubated for 1 h in mTALP at a concentration of 20 × 10^6^ sperm per mL in the presence of 0 (control), 0.5, 5, and 50 mM ectoine. Following this, sperm were exposed to hypoosmotic media (150 mOsm) for 15 min before assessing motility (CASA), viability (flow cytometry), and degree of tail folding. Bulls were assigned to categories of either high, medium, or low (n = 4 bulls per category) osmotic tolerance based on the sperm viability at 150 mOsm in the 0 mM ectoine treatment. Similarly, following preincubation with ectoine (0, 0.5, 5, and 50 mM), sperm were exposed to hyperosmotic media (400 mOsm) for 15 min before assessing motility (CASA) and viability (flow cytometry). Bulls were categorized as having high, medium, or low osmotic tolerance based on the sperm viability at 400 mOsm in control.

Experiment 4: effect of ectoine on sperm motility and kinematic parameters in cervicovaginal mucus

The objective of this experiment was to assess the protective ­effect of ectoine during incubation of sperm in cervicovaginal ­mucus. Mucus was collected from nulliparous beef heifers in spontaneous standing estrus, as described by Fernandez-Fuertes et al. (2016). The mucus from two cows was pooled and used throughout. Sperm (n = 6 bulls) were thawed, washed, and resuspended in mTALP at a final concentration of 50 × 10^6^ sperm/mL. Following this, sperm were incubated in the presence of 0 (control), 0.5, 5, and 50 mM of ectoine for 1 h, following which they were added to mucus to a final concentration of 10 × 10^6^ sperm per mL and incubated at 37 °C for 3 h. The presence of both cells and extracellular matter in the mucus made accurate assessment of motility and kinematics by CASA impossible. Therefore, motility (progressive, non-progressive, and immotile) was assessed subjectively using a negative phase contrast microscope (CX31; Olympus, USA) at a magnification of 200× fitted with a heated stage at 37 °C. A 10 µL droplet of sperm–mucus mixture was placed on a prewarmed slide, covered with a prewarmed coverslip, and evaluated by counting 100 sperm per sample across random fields in the center of the slide.

Computer assisted sperm analysis

Sperm samples were assessed using an IVOS II CASA system (IMV Technologies, L’Aigle, France), featuring a high-resolution internal camera with 1/1,000 s strobed illumination and 60 Hz frame rate, equipped with a Zeiss 10× negative phase-contrast objective (Zeiss 10x NH IVOS-II 160 nm) and temperature-regulated slide holder (37 °C). A 3 µL sample at 10 × 10^6^ sperm per mL in mTALP was loaded in a pre-warmed 20-micron Leja chamber (IMV Technologies), and a minimum of 400 sperm per sample were assessed in a minimum of eight fields of view. For sperm detection, in order to ensure consistent, reliable evaluations, recommended factory-programmed CASA settings for bull sperm were used throughout (O’Meara et al. 2022). Parameters assessed included: total motility (%), progressive motility (%), and kinematic parameters, including curvilinear velocity, (VCL; μm/s), straight line velocity (VSL; μm/s), average path velocity (VAP; μm/s), straightness (STR; %), linearity (LIN; %), and the amplitude of lateral head movement (ALH; μm), as defined by Donnellan et al. (2022). Fixed motility settings were as follows: static = VAP < 5 µm/s and VSL < 3 µm/s; slow = VAP < 20 µm/s and VSL < 2 µm/s; progressive = STR ≥ 60% and VAP ≥ 40 (µm/s). The cell size was between 6 and 70 µm^2^ and head minimum brightness was set to a minimum intensity of 190.

Flow cytometric assessment of sperm function

Sperm functional assessments were performed on a CytoFLEX flow cytometer from Beckman Coulter (Labplan, Dublin, Ireland) furnished with three different lasers with wavelengths of 405 nm (80 mW laser output, violet laser), 488 nm (50 mW laser output, blue laser), and 638 nm (50 mW laser output, red laser). Before each experiment, CytoFLEX was calibrated using quality control fluorospheres (Beckman Coulter). The sperm population was separated from debris and clumps with side and forward scatter characteristics. The positioning of this gating to detect the sperm population was verified by labeling a semen sample from a single reference bull (reference sample) with 1 µg/mL Hoechst 33342 (excited by 405 nm laser and detected with a 450/45 nm band-pass filter). For all assessments, 10,000 events at medium flow rate were recorded and analyzed in CytExpert software (Beckman Coulter; Version 2.3). For each parameter measured, the area of the signal pulse was used during data collation, and compensation was performed to correct spectral overlap.

Assessment of viability

Viability was assessed using two fluorescent probes: a nucleic acid probe, SYTO 16 (Invitrogen; Biosciences, Dublin, Ireland), and a non-viable cell probe, propidium iodide (PI; Life Technologies, Carlsbad, CA, USA). Sperm were diluted to a final concentration of 2 × 10^6^ sperm/mL in 250 μL mTALP. SYTO 16 was added at a final concentration of 100 nM and incubated at 37 °C in a heat block in the dark for 10 min. Subsequently, PI was added at a final concentration of 15 µM and incubated for a further 5 min. Post incubation, the SYTO 16 and PI were excited using a 488 nm laser and detected with a 525/40- and 690/50-nm band-pass filter, respectively, and the percentage of viable cells was expressed as the percentage of cells positive for SYTO 16 but negative for PI (Figure 1C).

Representative histograms and scatter plots obtained following the assessment of viability using live/dead dual staining. The areas of the histograms or plots highlighted by colored boxes identify the populations analyzed in this study. The blue-colored box (A) highlights a sperm subpopulation positive for SYTO 16. The highlighted population indicated by the green-colored box (B) identifies the sperm population negative for Propidium Iodide (PI). The bottom right quadrant, colored in red (C) on the scatter plot highlights all sperm that are PI-/SYTO16+ and therefore determined as viable.

Assessment of membrane fluidity and acrosome integrity

Acrosome integrity, membrane fluidity, and viability (as detected by membrane integrity) were assessed simultaneously using PNA-Alexafluor-647 (PNA-AF-647; Invitrogen), Merocyanine 540 (M540; Sigma Aldrich), and 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen), respectively. Sperm were diluted to a final concentration of 2 × 10^6^ sperm/mL in 250 μL mTALP. Samples were labelled with a final concentration of 0.5 µg/mL PNA-AF-647, 0.8 µM M540, and 3 µM DAPI. Samples were then incubated for 15 min at 37 °C prior to assessment. The PNA-AF-647, M540, and DAPI were ­excited using a 635, 488, and 405 nm laser, and detected with a 660/20-, 585/42- and 450/45-nm band-pass filters, respectively. The percentage of live cells with low membrane fluidity was expressed as the proportion of cells negative for both DAPI and M540 (Figure 2C), while the percentage of acrosome intact sperm was expressed as the proportion of sperm negative for PNA-AF-647 (Figure 2D).

Representative histograms and scatter plots illustrating the simultaneous evaluation of (A) viability (B, C) membrane fluidity and (D, E) acrosome integrity using 4′,6-diamidino-2-phenylindole (DAPI), merocyanine 540 (M540), and PNA–Alexa Fluor 647 (PNA-AF-647), respectively. Highlighted regions within the histograms and plots (green boxes) indicate the specific sperm populations analyzed. For membrane fluidity, the green box on the histogram (B) identifies sperm with low membrane fluidity, while the corresponding scatter plot (C) shows the percentage of sperm which are live and have low membrane fluidity. Similarly, for acrosome integrity, the green box on the histogram (D) denotes sperm with an intact acrosome, and the scatter plot (E) shows the percentage of sperm which are live and have an intact acrosome.

Statistical analysis

For all experiments, the bull was the experimental unit. Data were analyzed using the Statistical Package for Social Sciences (IBM SPSS for Windows 25, Armonk, N.Y., USA). In Experiments 1, 2, and 4, data were analyzed using a two-way repeated measures ANOVA combined with Bonferroni post-hoc tests. A test of sphericity was used prior to performing repeated measures ANOVA using Mauchly’s tests. In Experiments 3a and 3b, normality of distribution was assessed using the Shapiro-Wilk test following which a one-way univariate analysis of variance (ANOVA) was performed, followed by Bonferroni post hoc tests. The results were considered statistically significant if P < 0.05. All results are presented as mean ± SEM.

Results

Experiment 1: assessment of the effect of ectoine on in vitro sperm functional parameters

To examine the biocompatibility of ectoine with sperm, sperm motility and functional parameters were evaluated over a 6 h incubation period. All variables were found to vary across time, with the exception of ALH (Table 1; P > 0.05). Those variables that differed over time primarily decreased over the 6 h incubation period, a response that would be expected following an extended incubation of processed sperm. There was no treatment by time interaction for total motility, progressive motility, VCL, ALH, BCF, LIN, and WOB (P > 0.05). However, an interaction was evident for VSL, VAP, and STR (P < 0.05). In the control (0 mM), 0.5 mM, and 5 mM groups, VSL was maintained over the 6 h incubation period, whereas the 50 mM treatment exhibited a decline after 3 h of incubation (P < 0.001). VAP and STR showed a similar pattern, whereby the 50 mM ectoine treatment resulted in a continuous decline over 6 h, whilst all other ectoine treatments conserved these parameters over time (Table 1). Ectoine treatment had a significant effect on BCF, LIN, and WOB (P < 0.05), with the 50 mM concentration yielding lower values than all other treatment groups. This effect was independent of time (Table 1). In contrast, there was no effect of treatment on viability and acrosome integrity (Table 1; P > 0.05). Similarly, the proportion of viable sperm exhibiting low membrane fluidity was comparable between all treatments (P > 0.05).

Experiment 2: assessment of the protective effects of ectoine on heat stressed sperm

There was no treatment by time interaction for total motility at 39 °C but there was an effect of both time (P < 0.001) and treatment (Figure 3A; P < 0.01). Regardless of treatment, sperm motility declined over the 6 h incubation period (P < 0.05). There were no differences between sperm treated with 0.5 or 5 mM ectoine and the 0 mM control group, irrespective of incubation time (P > 0.05). In contrast, sperm treated with 50 mM ectoine exhibited lower overall motility compared to the control (Figure 3A; P < 0.01).

Effect of ectoine on bull sperm post-thaw during heat stress. Total motility of (A) sperm incubated at 39 °C and (B) sperm incubated at 42 °C and assessed at 0, 3, and 6 h (n = 8 bulls). abDifferent superscripts indicate a significant difference (P < 0.05). Data are displayed as mean ± SEM. ns = non-significant.

At 42 °C, similar to the effects of heat stress at 39 °C, total motility declined over the 6 h incubation period (P < 0.05). A treatment by time interaction was also evident at 42 °C (Figure 3B; *P *< 0.05). In the control group (0 mM), total motility declined between 0 and 3 h of incubation (P < 0.05), whereas sperm treated with 0.5 and 5 mM ectoine maintained their motility over the same period (P > 0.05). Notably, sperm treated with 50 mM ectoine exhibited motility comparable to all other treatments at 0 h; however, a decline in motility was observed by 3 h of incubation (P < 0.05). By 6 h, motility levels were similar across all treatments, likely reflecting the substantial decline in motility observed between 3 and 6 h.

Experiment 3: assessment of the protective effects of ectoine against osmotic stress

Experiment 3a: optimization of the solutions used for hypoosmotic and hyperosmotic media

Hypoosmotic conditionsAs the osmolarity was reduced from 300 mOsm to 50 mOsm, motility declined linearly (Supplementary Figure S1, A; P < 0.001), with a drop in motility evident at 150 mOsm compared to the control (P < 0.05). As osmolarity declined, the percentage of viable sperm remained similar to the control (Supplementary Figure S1, B; *P *> 0.05). Additionally, tail folding increased as osmolarity decreased (Supplementary Figure S1, C; P < 0.001), with 150 mOsm being the highest osmolarity at which a significant difference from the control was observed, and was therefore selected for subsequent experiments.Hyperosmotic conditionsAs the osmolarity was increased from 300 to 600 mOsm, the motility of sperm declined linearly (Supplementary Figure S2, A; P < 0.001), with an almost complete loss of motility observed at 500 mOsm. Sperm viability remained comparable to the control across all osmolarities tested (Supplementary Figure S2, B; P > 0.05). Given these observations, an osmolarity of 400 mOsm was selected as the threshold for all further hyperosmotic analyses.

Experiment 3b: effect of ectoine on protecting sperm against osmotic stress (low and high osmolarity)

Incubation of sperm in hypoosmotic media (150 mOsm) ­resulted in a reduction in total motility compared with isoosmotic ­conditions (300 mOsm) in bulls classified as having low osmotic tolerance (Figure  4A; P < 0.05). In contrast, no effect of osmotic challenge was observed in bulls with high osmotic tolerance. In the low osmotic tolerance group, preincubation with ectoine concentrations of 0.5 and 5 mM did not improve motility relative to the 0 mM control. However, sperm incubated with 50 mM ectoine exhibited higher motility compared with the control (Figure  4A; P < 0.05).

Effect of ectoine on protecting sperm against hypoosmotic conditions. (A) total motility and (B) viability of bull sperm incubated at 37 °C for 15 min in 150 mOsm mTALP following exposure to 0, 0.5, 5, and 50 mM ectoine (n = 12 bulls). Iso media was mTALP media of 300 mOsm osmolarity which acted as the control. Data are displayed as mean ± SEM. abcDifferent superscripts indicate a significant difference within osmotic tolerance groups (P < 0.05). Bulls were assigned to categories of either high, medium or low (n = 4 bulls per category) osmotic tolerance based on the sperm viability at 150 mOsm in the 0 mM ectoine treatment.

A similar pattern was observed for viability. Sperm viability was unaffected by hypoosmotic exposure in bulls with high osmotic tolerance (P > 0.05); however, viability decreased in the medium and low osmotic tolerance groups compared with isoosmotic conditions (Figure  4B; P < 0.05). Preincubation with 0.5 and 5 mM ectoine resulted in viability comparable to the 0 mM control, whereas incubation with 50 mM ectoine increased viability in the medium and low osmotic tolerance groups (Figure 4B; P < 0.05). Notably, the increased tail folding observed in the 50 mM ectoine group (Supplementary Figure S3; P < 0.001) is indicative of enhanced membrane integrity, consistent with the improved motility and viability observed under hypoosmotic conditions.

When assessed under hyperosmotic conditions, total motility decreased upon incubation of the sperm in hyperosmotic (400 mOsm) compared to isoosmotic (300 mOsm) media (P < 0.05). In contrast, viability remained constant across all treatments, with no difference observed between the isoosmotic control and sperm incubated in hyperosmotic conditions (P > 0.05). Addition of ectoine to sperm prior to hyperosmotic media exposure did not affect the total motility or viability compared to the no ectoine treatment in the high, medium, and low osmotic tolerance groups (Supplementary Figure S4; P > 0.05).

Experiment 4: effect of ectoine on sperm motility and kinematic parameters in cervicovaginal mucus

Both treatment and time had an effect on total motility (Figure 5; P < 0.01), however, there was no treatment by time interaction (P > 0.05). Across all treatments, total motility declined linearly over the 3 h incubation period in mucus. Sperm treated with 50 mM ectoine exhibited higher motility throughout incubation compared to the control (0 mM) (P < 0.05), with the difference becoming particularly evident after 3 h. There was a time by incubation medium (mucus versus mTALP) interaction (P < 0.05), where sperm incubated in cervicovaginal mucus experienced a 50% reduction in total motility over 3 h, whereas those incubated in mTALP retained motility. Treatment with 50 mM ectoine mitigated the effect of mucus incubation, maintaining motility at levels comparable to sperm incubated in mTALP (P > 0.05).

Total motility of frozen-thawed bull sperm in the absence or presence of ectoine, incubated for 3 h in oestrus phase cervicovaginal mucus (n = 6 bulls). Media control in mTALP was included. Sperm were assessed subjectively using a phase contrast microscope at a magnification of 200×. abcDifferent superscripts indicate a significant difference (P < 0.05). Data are displayed as mean ± SEM. ns = non-significant.

Discussion

Ectoine is well-documented for its protective effects in bacteria, single-cell organisms, and mammalian cells, yet its impact on sperm function has remained unexplored until now. Here, we demonstrate for the first time that ectoine is non-cytotoxic to sperm and can enhance resilience under various stress conditions. Analysis of sperm functional attributes revealed that ectoine did not impact sperm motility, viability, acrosome integrity, or membrane fluidity over an extended 6 h incubation at 37 °C. Under thermal stress (42 °C), ectoine at 0.5 and 5 mM sustained sperm motility compared to the control. Additionally, ectoine improved viability and motility in sperm with low osmotic tolerance and maintained motility of sperm during prolonged exposure to cervicovaginal mucus.

Flow cytometry and CASA assessments are well-established techniques for evaluating sperm quality and have been widely used to investigate the relationship of sperm in vitro parameters with fertility (Kutchy et al. 2019; Bernecic et al. 2021). In this study, these tools were employed to assess the biocompatibility of ectoine with sperm and to determine its potential impact on key functional parameters. Ectoine’s non-cytotoxic profile, as observed in this study, aligns with its non-toxic role in other mammalian cell studies (Buommino et al. 2005; Choi et al. 2018). While motility remained largely unaffected, a reduction in kinematic parameters, including VSL, VAP, and STR was observed at 50 mM, suggesting subtle alterations in sperm energy dynamics or flagellar mechanics at higher doses. However, the absence of significant differences in total and progressive motility across all treatments suggests that ectoine does not fundamentally impair sperm movement. The acrosome integrity, viability, and low membrane fluidity levels in the current study were in line with previous studies of bull sperm (Sellem et al. 2015; Bernecic et al. 2021) and levels remained stable across ectoine treatments. This indicates that ectoine does not compromise membrane integrity or cellular viability, even at high concentrations. Such findings align with prior research demonstrating ectoine’s ability to ­stabilize cell membranes and proteins without inducing cytotoxicity (Graf et al. 2008).

Sperm are sensitive to heat stress, with increased temperature impairing motility through decreased mitochondrial activity and ATP synthesis (Gong et al. 2017). Therefore, in this study, moderate and high heat stress (39 °C and 42 °C) was applied as a functional stress test to assess ectoine’s protective effects. Overall, upon exposure to thermal stress, ectoine had the capacity to modulate sperm motility, with subtle beneficial effects observed at 42 °C. Ectoine at 0.5 mM and 5 mM attenuated the decline in motility during the first 3 h, highlighting its potential as a thermal stress mitigator during short-term incubation. This is in line with studies displaying ectoine’s protective effect against heat stress in various organisms, such as the planktonic crustacean Daphnia magna (Adam et al. 2014), bacteria, such as E. coli (Malin and Lapidot 1996), enzymes (Lippert and Galinski 1992), and mammalian cells like keratinocytes (Buommino et al. 2005). Tanne et al. (2014) found that hydroxyectoine, an ectoine derivative, is even more effective at protecting against high temperatures, likely due to its higher glass transition temperature, stronger water-binding ability and superior protein-stabilizing properties (Smiatek et al. 2013; Salmannejad and Nafissi-Varcheh 2017).

The sperm plasma membrane is a highly specialized structure that plays a pivotal role in regulating key molecular processes essential for fertilization. Unlike somatic cells, sperm are transcriptionally and translationally inactive, rendering them incapable of repairing damaged membranes (Grunewald et al. 2005). Therefore, the stabilization of membrane proteins and lipid structures is crucial for preserving sperm function. The plasma membrane not only provides structural integrity and protection against mechanical and chemical stressors within the female reproductive tract but also governs ion homeostasis (e.g. Ca^2+^, K^+^, H^+^) and water flux, which are vital for sperm motility, capacitation, and the acrosome reaction, all processes necessary for successful fertilization. To assess the functional integrity of the sperm membrane, hypoosmotic swelling tests (HOST) have been developed to evaluate the sperm’s ability to regulate water movement under hypoosmotic conditions. This study revealed significant variability in osmotic tolerance among bulls. Despite showing comparable viability and total motility under isoosmotic conditions, bulls responded differently when exposed to osmotic stress. This suggests that baseline sperm quality metrics alone may not reliably predict a bull’s ability to withstand osmotic challenges. Sperm with compromised membrane functionality exhibited greater sensitivity to osmotic stress, leading to increased membrane damage, loss of motility, and reduced viability. Importantly, supplementation with 50 mM ectoine prior to hypoosmotic exposure significantly improved both sperm viability and total motility in bulls classified as having medium and low osmotic resistance, compared with untreated controls. These results suggest that ectoine exerts a stabilizing effect on sperm membranes, mitigating osmotic stress and preventing membrane lysis, consistent with ectoine’s established role in stabilizing cytoplasmic proteins, lipid bilayers, and membrane-associated proteins (Lippert and Galinski 1992; Harishchandra et al. 2010). Although protective effects were observed under hypoosmotic conditions, no significant effect was detected in hyperosmotic media. This is in contrast to reported protective effects of ectoine on corneal epithelial cells against hyperosmotic stress (Li et al. 2024).

In species that are vaginal depositors of semen, the cervix is the main barrier to sperm progression, with less than 0.1% of sperm deposited in the vagina estimated to get across the cervix (Harper 1982). This is due to a combination of a complex anatomy, attack of sperm by the female immune system but also the outward flow of viscous, negatively charged cervical mucus, which sperm must penetrate through to reach the uterus. In the present study, the effect of cervicovaginal mucus on sperm motility was examined, and the study revealed that sperm incubated in cervicovaginal mucus had a 50% reduction in motility over 3 h, while sperm incubated in mTALP (a more physiological medium for sperm) maintained motility. The significant decline in motility over time suggests that the mucus may act as a barrier that impedes sperm movement over in vitro incubation (Lee et al. 2021). This aligns with findings from other studies that have shown that cervical mucus, particularly in species like humans and cows, presents a challenging environment for sperm due to its varying viscosity, composition, and penetrability throughout the estrous cycle (Huang et al. 2024). Mucus was collected from heifers during the follicular phase of the cycle; however, it is important to note that the mucus used in this study originates from the vagina of the reproductive tract, containing secretions from both the cervical lumen and vaginal regions. In vivo, sperm traverse mucus within privileged pathways formed by cervical microgrooves rather than moving directly through the cervical lumen (Mullins and Saacke 1989). Therefore, the effects of these different sources of mucus on sperm function cannot be entirely ruled out. Regardless, this provided a challenging environment for sperm to examine ectoine’s protective potential. The results indicated that ectoine supplementation influenced sperm motility in cervicovaginal mucus, with 5 mM and 50 mM concentrations significantly maintaining motility within cervicovaginal mucus at 3 h compared to the control. In ocular studies which applied ectoine topically to a murine model of experimental dry eye disease, it was shown to increase mucin production, including secreted mucins MUC5AC and MUC2 (Lin et al. 2024). These mucins are primarily responsible for mucus hydration and viscosity in the female reproductive tract (Sheng and Hasnain 2022), suggesting ectoine may alter the mucin structure of cervicovaginal mucus, modifying the viscoelastic properties in a way that facilitates sperm progression.

This study provides novel insights into the biocompatibility and protective potential of ectoine on sperm function under various stress conditions. These findings demonstrate that ectoine is non-cytotoxic to sperm and can enhance their resilience when thermally or osmotically stressed as well as exposure to cervicovaginal mucus. The ability of ectoine to mitigate against a thermally induced decline in sperm motility at elevated temperatures aligns with its well-documented cytoprotective properties in other biological systems. Furthermore, its stabilizing effect on sperm membranes in hypoosmotic conditions suggests a role in preserving structural integrity, particularly in sperm with compromised osmotic tolerance. The observed maintenance of sperm motility in cervicovaginal mucus further underscores ectoine’s potential to improve sperm transport within the female reproductive tract. Collectively, these findings establish ectoine as a promising ­candidate for improving sperm resilience and warrant further studies to assess additional protective effects of ectoine and the practical applications of ectoine in assisted reproduction.

Supplementary Material

skag015_Supplementary_Data