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. 2025 Sep 26;60(9):e70126. doi: 10.1111/rda.70126

Evaluation of a Chemically Defined, Long‐Term Extender for Liquid Storage of Stallion Semen

Leonardo F C Brito 1,✉, Renata L Linardi 1, Leslie A S Rosales 1, Nithiya Sri Balamurugan 1, Camilo Hernández‐Avilés 2, Luisa Ramírez‐Agámez 2
PMCID: PMC12465434  PMID: 41002042

ABSTRACT

Efficient use of stallion semen in liquid state is limited by its relatively short shelf‐life. A chemically defined extender (Beyond) is now available for long‐term liquid semen preservation. The objectives of the present study were to compare Beyond with milk extenders for the preservation of semen at two temperatures, and to evaluate fertility of semen cooled for 4–8 days before artificial insemination. Semen was processed using different extenders: milk, cholesterol (BotuSemen Special); milk‐based (INRA 96); and Beyond. Sperm motility, membrane and acrosome integrity, and chromatin structure were evaluated in semen stored at 17°C for 7 days or at 5°C for 14 days. Sperm motility decreased in the first few days of storage regardless of extender or storage temperature. Sperm motility continued to decline at relatively constant rates in semen extended in milk extenders, but the rate of decline was substantially reduced with Beyond. Sperm motility in semen extended with Beyond was greater than in semen extended with milk extenders after 4 days of storage at 17°C, or after 7 days of storage at 5°C. Extender did not affect sperm DNA damage during storage, but sperm with intact membrane and intact acrosome were lower with Beyond. Inseminations with semen stored with Beyond at 5°C for an average of 5.5 days resulted in embryos in 61% of cycles (11/18). In conclusion, Beyond extender resulted in greater sperm motility longevity when compared to milk extenders, especially when semen was stored at 5°C. Satisfactory fertility was obtained with semen cooled for 4–8 days before artificial insemination.

Keywords: artificial insemination, extender, semen, sperm, stallion

1. Introduction

Transported, liquid stallion semen has been routinely used for artificial insemination (AI) for almost 40 years. Nonetheless, the fertility potential of stored liquid semen is maintained, at best, for 72 h after semen collection. The longest period of liquid semen storage resulting in pregnancies reported in a peer‐reviewed study was 80 h (Heiskanen et al. 1994). Hence, most equine breeding programs are designed to use semen within 48 h after collection (Aurich 2008; Clulow and Gibb 2022; Katila 1997). Reported pregnancy rates obtained with cooled semen in smaller controlled studies are usually greater than 60% and sometimes as high as 90% (Douglas‐Hamilton et al. 1984; Shore et al. 1998; Varner et al. 1989). However, most larger studies involving commercial breeding programs using semen stored for 6–24 h in different conditions report pregnancy rates ranging from 45% to 65% (Cuervo‐Arango et al. 2015; Kareskoski et al. 2019; Newcombe and Cuervo‐Arango 2011; Sieme et al. 2003; Tanner and Barrell 2024).

Semen extenders are crucial for sperm preservation in liquid state. For stallions, most extenders are based on a combination of skim milk solids supplemented with glucose and antibiotics (Kenney et al. 1976; Varner et al. 1989). Commercial milk‐based extenders contain proprietary extracts of milk phosphocaseinate, which is the major milk component that protects sperm quality during cooled storage (Batellier et al. 2001). Commercial extenders have also been modified and optimised to include different buffers, antioxidants and energy substrates (Aurich 2005; Clulow and Gibb 2022). Although some studies report the use of semen stored at room temperature (Katila 1997), cooling results in decreased metabolic demand and helps preserve sperm viability (Gibb and Aitken 2016; Varner et al. 1989). Beyond is a commercial, chemically defined extender that purportedly does not contain animal by‐products. In addition, the extender is marketed for long‐term storage of stallion semen. According to the manufacturer, semen processed with the extender can be preserved for 7 days at 17°C, or for up to 14 days at 5°C. The formulation of the Beyond extender is proprietary.

The ability to preserve sperm fertilising potential for periods longer than 72 h, as well as to store semen at temperatures closer to room temperature, might facilitate the logistics of equine breeding programmes using liquid semen. The objectives of the present studies were to (1) evaluate the effect of temperature (17°C or 5°C) on sperm preserved with Beyond extender compared to other commercial milk and milk‐based extenders and (2) evaluate fertility of stallion cooled semen processed with Beyond extender and stored at 5°C for 4–8 days. We hypothesised that sperm motility, membrane and acrosome integrity, and chromatin structure differ in semen extended with a new chemically defined, long‐term extender when compared to existing milk and milk‐based extenders.

2. Materials and Methods

2.1. Animals

All procedures involving live animals were performed according to the United States Government Principles for the Utilisation and Care of Vertebrate Animals Used in Testing, Research and Training and were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania (Protocol # 806502). Informed consent was obtained from client‐owned horses. The study was conducted between January and August in the Northern hemisphere, and all stallions were healthy and known to produce semen of reasonable quality (≥ 65% motile sperm and ≥ 35% progressively motile sperm).

2.2. Experiment I

The first experiment was conducted to determine technical aspects related to the evaluation of sperm motility during long‐term storage in semen preserved with Beyond extender (Minitube, Verona, WI, USA). Due to concerns related to the possible depletion of energy substrate during prolonged storage, semen samples were diluted with fresh extender and incubated prior to analysis. The objective of this experiment was to determine the effects of the extender used for dilution on sperm motility using a split‐sample design (see Figure S1 for experimental design diagram). Six ejaculates were obtained from three stallions (1 American Quarter Horse, 2 Arabian; 8–20 year‐old; 2 ejaculates/stallion). Semen was processed using Beyond extender and sperm motility was evaluated daily in semen stored at 5°C for 7 days after dilution with either Beyond extender or EquiPlus extender (Minitube).

2.3. Experiment II

The objective of this experiment was to determine the effects of extender and storage temperature on sperm quality during long‐term storage using a split‐ejaculate, factorial design (see Figure S2 for experimental design diagram). Twelve ejaculates were obtained from six stallions (2 American Quarter Horse, 2 Arabian, 1 Thoroughbred, 1 Trakehner; 8–20 year‐old; 2 ejaculates/stallion). Semen was processed using commercial extenders with different formulations: milk, cholesterol (BotuSemen Special extender; BotuPharma, Phoenix, AZ, USA); milk‐based (INRA 96 extender; IMV Technologies; Maple Grove, MN, USA); and chemically defined, long‐term (Beyond extender). Sperm motility was evaluated daily in semen stored at 17°C for 7 days or at 5°C for 14 days. In addition, sperm membrane integrity and fluidity, acrosome status, and chromatin structure were evaluated using flow cytometry after 2, 7 and 14 days of storage.

2.4. Experiment III

This experiment was designed as a proof of concept that semen processed using Beyond extender remains fertile after 4–8 days of cooled storage. Twenty‐three ejaculates were obtained from two stallions (American Quarter Horse and Arabian; 8–9 year‐old; 10–13 ejaculates/stallion). Sperm motility was evaluated daily in semen stored at 5°C for up to 14 days. Semen doses stored between 4 and 8 days were used for AI. Semen doses were produced ahead of time and used as needed according to the mare's breeding management plan; therefore, the number of inseminations was well distributed between stallions and across storage days. Seven mares (Standardbred and Thoroughbred; 14–21 year‐old) were used to perform 18 inseminations (1–4 estrous cycles/mare).

2.5. Semen Processing

Semen was obtained using a Missouri‐style artificial vagina with the stallion mounted on a dummy. Immediately after collection, semen was filtered to remove gel, and the ejaculate volume was inferred by weight. Sperm concentration was determined using a NucleoCounter SP100 (ChemoMetec, Allerod, Denmark). Sperm morphology was determined by examining 100 sperm using formalin‐fixed wet preparations evaluated under 1000× magnification and immersion oil using differential interference contrast (DIC) microscopy. Sperm motility was evaluated as described below, and only samples with ≥ 35% progressively motile sperm were processed further. Semen samples were immediately extended (1:1, v/v) with pre‐warmed (38°C) EquiPlus extender with Amikacin and Penicillin (Minitube), allocated into 50‐mL conical tubes and allowed to equilibrate at room temperature (20°C–22°C) for 30 min. Then, 1 mL of cushion fluid (MaxiFreeze; IMV Technologies) was layered beneath the extended semen using an Argyle tomcat catheter (3.5 Fr, 14 cm; Covidien, Mansfield, MA, USA), and tubes were centrifuged at 1000×g for 20 min. After centrifugation, the cushion was removed using a tomcat catheter, and the supernatant was carefully removed using a Pasteur pipette attached to a siphoning apparatus. Sperm pellets were resuspended at room temperature to a final concentration of 50 × 106 sperm/mL using the extender tested. Split samples stored at 17°C (range 17°C–19°C) were kept in a controlled‐temperature benchtop incubator (H2200‐HC MyTemp Mini Incubator; Stellar Scientific, Baltimore, MD, USA) for 7 days. Split samples designated for 5°C storage were initially kept in an Equitainer (Hamilton Biovet, Ipswich, MA, USA) for 24 h before being transferred into a fridge at 5°C (range 4°C–7°C) for storage for 14 days.

2.6. Sperm Motility Analysis

Sperm motility analysis was performed daily using a computer‐assisted sperm analysis (CASA) system (HT IVOS II; Hamilton Thorne Research, Beverly, MA, USA). Semen samples were thoroughly mixed, diluted to 20 × 106/mL with pre‐warmed extender, and incubated at 38°C for 10 min before evaluation. Pre‐centrifugation semen samples were diluted into EquiPlus extender before evaluation. Based on results from Experiment I (see below), samples processed in Beyond were also diluted into EquiPlus extender, whereas samples processed in INRA 96 and BotuSemen Special were diluted into the same extenders before evaluation. After incubation, 3 μL of extended semen was loaded into the chamber of a pre‐warmed 20‐μm Leja slide (IMV Technologies). A total of six fields and 300 sperm were evaluated. Videos were acquired at a frame rate of 60 Hz, and 45 frames were analysed. Sperm detection parameters included 170 minimum brightness, 4–54 μ2 area, and 10%–100% elongation. Parameters evaluated included percent total motility, percent progressive motility, beat cross frequency (BCF), curvilinear velocity (VCL), average path velocity (VAP) and straight‐line velocity (VSL). Sperm were considered static when VAP < 4 μm/s. Sperm were considered progressive motile when VAP > 30 μm/s and ([VSL/VAP] × 100) > 50%.

2.7. Sperm Plasma Membrane and Acrosome Status

In addition to the day of initial semen processing, samples stored at 17°C and 5°C were evaluated after 2 and 7 days of storage. Samples stored at 5°C were also evaluated after 14 days of storage. Sperm plasma membrane (plasmalemma) and acrosome membrane were evaluated using a 4‐laser flow cytometer (Cytoflex S Benchtop Flow Cytometer and CytExpert Software; Beckman Coulter, Brea, CA, USA). For plasma membrane integrity, SYBR‐14 fluorescence was detected using a 488‐nm laser and a 524/40 BP filter and propidium iodide (PI) fluorescence was detected using a 561‐nm laser and a 585/42 BP filter. For acrosome status, FITC‐conjugated peanut agglutinin (FITC‐PNA) fluorescence was detected using a 488‐nm laser and a 524/40 BP filter and PI, as described above. For sperm plasma fluidity, YO‐PRO‐1 fluorescence was detected using a 488‐nm laser and a 525/40 BP filter, merocyanine 540 fluorescence was detected using a 561‐nm laser and a 585/42 BP filter and Hoechst 33342 fluorescence was detected using a 405‐nm laser and a 450/45 BP filter. Unless otherwise stated, fluorophores were obtained from Molecular Probes (ThermoFisher, Eugene, OR, USA).

For analysis, 60 × 103 sperm were added to 500 μL of phosphate‐buffered saline (PBS) and incubated with the appropriate fluorophores at 37°C in the dark for 10 min prior to evaluation. The LIVE/DEAD Sperm Viability Kit containing SYBR‐14 (final concentration 0.5 nM) and propidium iodide (PI; final concentration 60 nM) was used to evaluate sperm viability. Acrosome integrity was evaluated with FITC‐PNA (Sigma‐Aldrich, Saint Louis, MO, USA) with a final concentration of 1 ng/mL, and PI with a final concentration of 37.5 nM. For the evaluation of sperm plasma membrane fluidity, 60 × 103 sperm were added to 1 mL of PBS and incubated at 37°C in the dark for 10 min with YO‐PRO‐1 (final concentration 25 nM), merocyanine 540 (final concentration 2.6 μM), and Hoechst 33342 (final concentration 0.75 μg/mL).

After staining, 200 μL of the sperm suspension was added to a microwell plate for analysis. The sperm population was initially gated based on forward (FSC) and side scatter (SSC) plots. For each analysis, 10,000 events in the gated region were acquired and plotted on graphs for the log fluorescence of the fluorophores used for evaluation. Scatter plots were gated to determine the percentage of viable sperm (SYBR‐14 positive), acrosome‐intact sperm (FITC‐PNA negative) and dead sperm (PI positive). Low, medium and high merocyanine staining were evaluated in viable sperm (low YO‐PRO staining) using fluorescence scatter plots that also showed medium and high YO‐PRO staining.

2.8. Sperm Chromatin Structure Assay (SCSA)

Samples stored at 17°C and at 5°C were frozen at −80°C after 0, 2 and 7 days of storage. In addition, samples stored at 5°C were also frozen at −80°C after 14 days of storage. Due to transport logistical challenges, the samples thawed during transit to the laboratory performing the analysis and were refrozen at −80°C before evaluation. The SCSA was performed using flow cytometry (FACScan, Becton Dickinson, Mountain View, CA) as previously described (Love and Kenney 1998). The instrument was equipped with a 488 nm argon laser at 20 mW and fluorescent detectors (bandpass 530/30 nm and long pass 670 nm). Each sample was thawed at 37°C in a water bath, and 20 μL of thawed semen was added to 200 μL of buffer solution (TNE buffer; 0.186 g disodium EDTA, 0.790 g Tris–HCl, 4.380 g NaCl in 500 mL deionised water, pH 7.4), mixed with 400 μL acid‐detergent solution (2.19 g NaCl, 1.0 mL 2 N HCl solution, 0.25 mL Triton‐X100, q.s. 250 mL deionised water), and held on ice for 30 s. Then, 1.2 mL acridine orange solution (3.8869 g citric acid monohydrate, 8.9429 g Na2HPO4, 4.3850 g NaCl, 0.1700 g disodium EDTA, 4 μg/mL acridine orange stock solution [1 mg/mL], deionised water q.s. 500 mL; pH 6.0) was added.

The sample was immediately placed in the flow cytometer and ran for 30 s to allow equilibration, after which the analysis was performed. The sperm population was initially gated based on forward (FSC) and side scatter (SSC) linear plots. For the analysis, 5000 events in the gated region were acquired. Data were stored in List mode and analysed using WinList software (Verity Software House; Topsham, ME, USA). Histograms of green versus red fluorescence were used to determine the percentage of cells outside the main population (COMPαt), which represents the sperm population with increased red fluorescence and, thus, DNA damage susceptibility. A sample from a known control stallion was used as a biologic control to standardise instrument settings each day of use.

2.9. Breeding Trial

Mares were teased to a stallion for estrus detection, and the ovaries and uterus were examined by transrectal palpation and ultrasonography (B‐mode, 7.5 MHz linear probe) once estrus was detected. When an ovarian follicle ≥ 35 mm was detected concomitantly with more than 48 h of endometrial edema ≥ 2 (0–3 scale) and/or 2 days of estrus, 1.8 mg deslorelin (Sucromate; Thorn BioScience, Louisville, KY, USA) was administered intramuscularly. Artificial insemination was performed into the uterine body either at the time of deslorelin treatment or 24 h after treatment. Mares were examined between 4 and 24 h after insemination for signs of persistent post‐breeding endometritis, and therapy (uterine lavage and oxytocin treatments) was instituted as needed. Daily exams were performed until ovulation was detected and the uterus was clear of any fluid. Pregnancy diagnosis was performed by transrectal ultrasonography 12–15 days after ovulation. Pregnancy was terminated 15 days after ovulation by administering 250 μg cloprostenol (Estrumate; Merck Animal Health, Rahway, NJ, USA) intramuscularly. For one mare/cycle, the uterus was lavaged 7 days after ovulation to recover an embryo. Fertility was defined by the observation of an embryo in utero or after uterine lavage.

2.10. Statistical Analysis

Statistical analysis was conducted using GraphPad Prism V. 9.4.0 (GraphPad Software, San Diego, CA, USA). Normal distribution was evaluated using Residual QQ Plots, and residuals mostly followed a straight line. In Experiment I, the effect of the extender used for semen dilution prior to sperm motility analysis was determined using a Paired t‐test. In Experiment II, the main effects of extender, time and extender‐by‐time interaction were determined using a Mixed‐effects model with ejaculate included as a random effect. The model used a compound symmetry covariance matrix and was fit using Restricted Maximum Likelihood (REML). Tukey's test was used for post hoc comparisons when main effects were observed. In Experiment III, the main effects of stallion, time and stallion‐by‐time interaction were determined using a Mixed‐effects model with ejaculate included as a random effect. Differences in the characteristics of cooled semen doses that resulted in embryos (fertile) and those that did not result in embryos (non‐fertile) were determined using one‐way ANOVA.

3. Results

3.1. Experiment I

Results of initial semen analysis are included in Table S1. When evaluating semen processed using Beyond extender, there were small but significant differences between Beyond and EquiPlus extenders when used to dilute semen immediately before sperm motility evaluation. Use of Beyond extender for dilution resulted in lower (p < 0.005) percentages of motile sperm (mean difference: −3.3%, 95% CI: −5.1% to −1.4%) and progressively motile sperm (mean difference: −5.5%, 95% CI: −7.4% to −3.6%) when compared to EquiPlus extender (Figure S3). Based on these results, semen extended with Beyond extender was diluted with EquiPlus before sperm motility evaluation in Experiments II and III.

3.2. Experiment II

Results of initial semen analysis are included in Table S2. The final concentration for semen extended in BotuSemen Special, INRA 96 and Beyond was 50.4 ± 0.6, 51.6 ± 0.5 and 50.8 ± 0.6 × 106 sperm/mL, respectively (mean ± SEM).

3.2.1. Storage at 17°C

There were extender, time and extender‐by‐time effects (p < 0.05) on motile and progressively motile sperm for semen stored at 17°C for 7 days (Figure 1). The percentage of motile sperm decreased (p < 0.05) after initial semen processing and final extension, and after 24 h of storage regardless of extender. The percentage of progressively motile sperm decreased (p < 0.05) after initial semen processing and final extension when Beyond extender was used, and after 24 h of storage regardless of extender. Motile and progressively motile sperm in semen extended with BotuSemen Special decreased rapidly and were lower (p < 0.05) than those in semen extended with either INRA 96 or Beyond after 2 days of storage. The rate of motility decline remained mostly constant until the end of the experiment in semen extended with INRA 96, but a reduced rate of decline in semen extended with Beyond was observed. As a result, the percentages of motile and progressively motile sperm in semen extended with Beyond were greater (p < 0.05) than in semen extended with INRA 96 after 4 days of storage.

FIGURE 1.

FIGURE 1

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Mean (±SEM) percentages of motile sperm (top) and progressive motile sperm (bottom) for stallion semen (n = 12 ejaculates, 6 stallions) stored at 17°C for 7 days (left) or at 5°C for 14 days (right). Semen samples were extended with commercial extenders with different formulation: milk, cholesterol (BotuSemen Special); milk‐based (INRA 96); and chemically defined, long‐term (Beyond). Day 0 refers to the initial ejaculate analysis, whereas Day 0′ refers to the sample after processing and final extension. BCF, beat cross frequency; STR, straightness; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight line velocity. Superscripts (*) indicate extender differences (p < 0.05) within day (*Beyond differs from BotuSemen Special and INRA 96, **Beyond differs from INRA 96, ***Beyond differs from BotuSemen Special, ****Beyond and INRA 96 differ from BotuSemen Special, *****All extenders differ). Superscripts (★) indicate time differences within extender from Day 0 or previous significant day (p < 0.05).

There were also extender, time and extender‐by‐time effects (p < 0.05) on BCF, VCL, VAP and VSL within both motile and progressively motile sperm populations (Figure 2, Figure S4). Beat cross frequency, VAP and VSL decreased (p < 0.05) after initial semen processing and final extension, and after 24 h of storage in semen extended with BotuSemen Special and INRA 96. In contrast, BCF, VCL and VAP did not decrease during storage in semen extended with Beyond, and the VSL rate of decline was reduced. As a result, BCF, VCL, VAP and VSL in semen extended with Beyond were greater (p < 0.05) than in semen extended with BotuSemen Special or INRA 96 after 2–3 days of storage. Bacterial contamination, as evidenced by observation of bacteria during sperm motility analysis or coagulation of the sample, was observed in four samples after 6 days of storage at 17°C (2 INRA, 1 BotuSemen Special, 1 Beyond); no sperm motility results were obtained from these samples.

FIGURE 2.

FIGURE 2

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Mean (±SEM) motile sperm kinetics for stallion semen (n = 12 ejaculates, 6 stallions) stored at 17°C for 7 days (left) or at 5°C for 14 days (right). Semen samples were extended with commercial extenders with different formulations: milk, cholesterol (BotuSemen Special); milk‐based (INRA 96); and chemically defined, long‐term (Beyond). Day 0 refers to the initial ejaculate analysis, whereas Day 0′ refers to the sample after processing and final extension. BCF, beat cross frequency; STR, straightness; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight line velocity. Superscripts (*) indicate extender differences (p < 0.05) within day (*Beyond differs from BotuSemen Special and INRA 96, **Beyond differs from BotuSemen Special, ***Beyond differs from INRA 96, ****Beyond differs from BotuSemen Special, *****All extenders differ). Superscripts (★) indicate time differences within extender from Day 0 or previous significant day (p < 0.05).

Representative histograms from flow cytometry analyses are included as Figure S5. There were extender (p < 0.05) and time effects (p < 0.01) on the percentages of sperm with intact membranes (SYBR+/PI‐) and sperm with intact acrosome (FITC PNA‐/PI‐). There was also a time effect (p < 0.0001) on the percentage of sperm with damaged DNA (COMPαt) (Figure 3). Percentages of sperm with intact membrane and intact acrosome were lower (p < 0.05) with Beyond than with BotuSemen Special or INRA 96 extenders. The percentage of sperm with intact membrane decreased (p < 0.05) after storage for 7 days, whereas sperm with intact acrosome decreased (p < 0.05) throughout the entire storage period. The percentage of sperm with damaged DNA increased (p < 0.001) throughout the entire storage period. There were time effects (p < 0.05) on the percentages of YO‐PRO‐negative sperm within all merocyanine staining intensity categories (Figure 4). There was also an extender‐by‐time effect (p < 0.05) on the percentage of sperm with low merocyanine staining. Most sperm showed low staining and the percentage of sperm within this category decreased (p < 0.05) after storage for 7 days with BotuSemen Special and INRA 96 extenders, but not with Beyond. The percentages of sperm with intermediate and high staining intensity increased (p < 0.05) during storage, but these were generally < 10% of the overall population. There was a time effect (p < 0.01) on the percentage of sperm with high YO‐PRO staining intensity, as that increased (p < 0.05) after storage for 7 days.

FIGURE 3.

FIGURE 3

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Mean (±SEM) percentage of SYBR‐positive/PI‐negative (intact membrane, top), FITC PNA‐negative/PI‐negative (intact membrane and acrosome, middle) and cells outside the main population (COMP) with acridine orange stainining (abnormal chromatin, bottom) for stallion semen (n = 12 ejaculates, 6 stallions) stored at 17°C for 7 days (left) or at 5°C for 14 days (right). Semen samples were extended with commercial extenders with different formulations: milk, cholesterol (BotuSemen Special); milk‐based (INRA 96); and chemically defined, long‐term (Beyond). Superscripts (★) indicate time differences within extender from Day 0 or previous significant day (p < 0.05).

FIGURE 4.

FIGURE 4

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Mean percentage of different sperm subpopulations according to merocyanine 540/YO‐PRO‐1 staining for stallion semen (n = 12 ejaculates, 6 stallions) stored at 17°C for 7 days (top) or at 5°C for 14 days (bottom). Semen samples were extended with commercial extenders with different formulations: milk, cholesterol (BotuSemen Special); milk‐based (INRA 96); and chemically defined, long‐term (Beyond).

3.2.2. Storage at 5°C

There were extender, time and extender‐by‐time effects (p < 0.05) on motile sperm, and time and extender‐by‐time effects (p < 0.0001) on progressively motile sperm for stallion semen stored at 5°C for 14 days (Figure 1). The percentages of motile and progressively motile sperm decreased (p < 0.05) after 24 h of storage in semen extended with BotuSemen Special and Beyond, but only declined significantly (p < 0.05) after 2 days in semen extended with INRA 96. The rate of motility decline remained mostly constant until the end of the experiment in semen extended with BotuSemen and INRA 96. The rate of decline in the percentage of motile sperm in semen extended with Beyond was initially similar to BotuSemen Special and INRA 96, but no further decline in motility was observed after 5 days of storage. In contrast, semen extended with Beyond had a marked decline in progressive motility after 24 h of storage that resulted in lower results (p < 0.05) than the other extenders for the first 3 days of storage. The rate of decline was reduced after 24 h and no further decline in progressive motility was observed after 5 days of storage. As a result, the percentages of motile and progressively motile sperm in semen extended with Beyond were greater (p < 0.05) than in semen extended with BotuSemen and INRA 96 after 7 days of storage.

There were also extender, time and extender‐by‐time effects (p < 0.05) on BCF, VCL, VAP and VSL within both motile and progressively motile sperm populations (Figure 2, Figure S4). Beat cross frequency, VAP and VSL decreased (p < 0.05) after 24 h of storage in semen extended with BotuSemen and INRA 96; most continued to decrease further during storage. In contrast, BCF, VCL, VAP and VCL did not decrease during storage in semen extended with Beyond. As a result, BCF, VCL, VAP and VSL in semen extended with Beyond were greater (p < 0.05) than in semen extended with BotuSemen or INRA 96 after 1–2 days of storage.

There were extender (p < 0.05) and time effects (p < 0.001) on the percentages of sperm with intact membranes (SYBR+/PI−), sperm with intact acrosome (FITC PNA−/PI−), and sperm with damaged DNA (COMPαt) (Figure 3). Percentages of sperm with intact membranes and intact acrosome were lower (p < 0.05) with Beyond than with BotuSemen Special or INRA 96 extenders. The percentage of sperm with damaged DNA was greater (p < 0.05) with BotuSemen Special than with INRA 96 extender. The percentage of sperm with intact membranes decreased (p < 0.05) after storage for 7 days and again after 14 days. The percentage of sperm with intact acrosome decreased (p < 0.05), whereas the percentage of sperm with damaged DNA increased (p < 0.001) after storage for 7 days. There were time effects (p < 0.05) on the percentages of YO‐PRO‐negative sperm within all merocyanine staining intensity categories (Figure 4). Most sperm showed low merocyanine staining, and the percentage of sperm within this category decreased during storage. The percentages of sperm with intermediate and high staining intensity increased (p < 0.05) after storage for 14 days, but these were generally < 10% of the overall population. There were extender (p < 0.05) and time (p < 0.001) effects on the percentage of sperm with intermediate YO‐PRO staining intensity. Overall, the percentage of sperm with intermediate YO‐PRO staining increased during storage and was lower with Beyond extender. There were time (p < 0.001) and extender‐by‐time (p < 0.05) effects on the percentage of sperm with high YO‐PRO staining intensity. Whereas sperm with high YO‐PRO staining increased (p < 0.05) after storage for 14 days with INRA 96 and Beyond extenders, no significant changes were observed with BotuSemen Special.

3.3. Experiment III

Results of initial semen analysis are included in Tables S3 and S4. There were stallion and time effects (p < 0.01) on motile sperm, and a time effect (p < 0.0001) on progressively motile sperm (Figure 5). Overall, the percentage of motile sperm was greater (p < 0.05) in Stallion 2 than in Stallion 1. The percentages of motile and progressively motile sperm decreased (p < 0.05) after initial semen processing and final extension, and after 24 h of storage. Motility did not decline significantly again until 8–10 days of storage.

FIGURE 5.

FIGURE 5

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Mean (±SEM) percentages of motile sperm (top) and progressive motile sperm (bottom) for stallion semen (Stallion 1, n = 10 ejaculates; Stallion 2, n = 13 ejaculates) extended with a chemically defined, long‐term extender (Beyond) and stored at 5°C for 14 days. Day 0 refers to the initial ejaculate analysis, whereas Day 0′ refers to the sample after processing and final extension. Superscripts (♦) indicate time differences from Day 0 or previous significant day across stallions (p < 0.05).

Results of all inseminations are included in Table S5. A single ovulation was detected within 24 h after insemination in 12 cycles and within 48 h in 5 cycles. Double ovulations were both detected within 48 h after insemination in one remaining cycle. Eleven inseminations (61%, 11/18) produced an embryo. These included 10 pregnancies that progressed normally until at least 15 days after ovulation and one Grade I blastocyst recovered 7 days after ovulation. A twin pregnancy was observed resulting from the double ovulation. Fertility did not seem to differ between stallions: 56% (5/9) and 67% (6/9) for Stallions 1 and 2, respectively. Semen storage lenght, interval between insemination and ovulation, sperm motility and morphology, total sperm, total motile sperm, and total morphologically normal, progressively motile sperm at the time of insemination did not differ (p > 0.05) between non‐fertile and fertile AI doses (Table 1).

TABLE 1.

Characteristics (mean ± SEM) of semen doses (2 stallions, 9 inseminations/stallion) prepared with a chemically defined, long‐term extender (Beyond) on the day of artificial insemination.

Non‐fertile (n = 7) Fertile (n = 11)
Semen storage length (days) 5.4 ± 0.5 5.5 ± 0.5
Ovulation detection after AI (h) 30.9 ± 4.4 32.7 ± 3.7
Total sperm motility (%) 71.8 ± 1.6 71.9 ± 2.1
Progressive sperm motility (%) 34.8 ± 2.1 35.6 ± 1.7
Normal sperm morphology (%) 71.1 ± 2.9 74.7 ± 1.7
Sperm concentration (×106/mL) 51.0 ± 0.6 52.0 ± 0.6
Total sperm (×106) 2547 ± 30 2599 ± 31
Total motile sperm (×106) 1830 ± 43 1872 ± 65
Total progressively motile sperm (×106) 885 ± 52 926 ± 45
Total morphologically normal, progressively motile sperm (×106) 638 ± 61 689 ± 32
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Note: Semen doses were stored at 5°C until insemination. Non‐fertile and fertile semen doses did not differ.

4. Discussion

Artificial insemination with cooled semen has become common in the equine industry. Still, satisfactory pregnancy rates (i.e., > 50% per‐cycle pregnancy rates) are generally only obtained with semen used within 48 h after collection; pregnancy rates decline rapidly if semen is stored longer before insemination (Aurich 2005; Clulow and Gibb 2022; Katila 1997). In this study, the breeding trial was designed as proof of concept since no peer‐reviewed study has reported the use of stallion cooled semen after more than 3.5 days. Researchers in Finland reported the use of semen stored for up to 80 h (Heiskanen et al. 1994), but all other studies in the literature report using semen stored for shorter periods of time. Hence, the breeding trial was designed so that only semen that had been stored 4–8 days was used for insemination. The fertilisation rate (61%) obtained with semen stored on average for 5.5 days was comparable to that reported in commercial breeding programmes. Despite the limitations of a small breeding trial, the results obtained with the chemically defined, long‐term extender tested herein are encouraging, as increasing the longevity (shelf life) of stallion cooled semen to 8 days post collection could facilitate the logistics of equine AI programmes.

Although the fertility observed with the chemically defined, long‐term extender in the present study (61%) was lower than that previously reported with semen processed with milk extenders and stored for up to 48 h at 5°C prior to insemination in small breeding trials (Douglas‐Hamilton et al. 1984; Shore et al. 1998; Varner et al. 1989), it was similar to pregnancy rates reported with milk‐based extenders (Batellier et al. 2001), and to those from commercial breeding programmes (Cuervo‐Arango et al. 2015; Kareskoski et al. 2019; Sieme et al. 2003). Breeding programmes with Norwegian Coldblooded trotters in Norway (Haadem et al. 2015) and Standardbreds in New Zealand (Tanner and Barrell 2024) have shown significantly lower pregnancy rates with cooled semen (approximately 45%–55%) when compared with fresh semen (60%–65%) when these semen types were used contemporaneously. In the UK, the reported per‐cycle pregnancy rate was 59% for mares of various breeds inseminated at different intervals from ovulation (Newcombe and Cuervo‐Arango 2011). Limitations of the present study included a small number of inseminations and the consequent narrow range of variation within the study population. When semen quality was considered, the authors would have placed the two stallions used in the breeding trial within the top quartile of the population. On the other hand, the mares used in the trial were older, susceptible mares that required close management and intense post‐breeding intervention. Advanced age and reproductive status of mares may have influenced fertility outcomes.

The long‐term extender ability to preserve sperm fertility seemed to be associated with the extender effects on sperm metabolism. The percentage of motile sperm was initially actually lower with Beyond extender, but reduced rates of decline in sperm motility were observed when compared to the other extenders. As a result, when compared to milk and milk‐based extenders, the percentage of motile sperm was greater after 4 days at 17°C and after 7 days at 5°C when the long‐term extender was used. The percentage of progressively motile sperm was considerably lower than the percentage of motile sperm with Beyond extender, as a larger number of sperm with circular tracks seemed to be observed with that extender. These observations also suggest that the benefits of this extender over milk extenders might only be captured if semen is used after 72 h post‐collection, that is, true long‐term storage (≥ 96 h). The effects of the long‐term extender on sperm metabolism were strikingly highlighted when sperm kinetics were evaluated. While sperm activity (BCF) and speed (VCL, VAP, VSL) within motile and progressively motile sperm subpopulations decreased significantly over time with BotuSemen Special and INRA 96 extenders, these parameters practically did not change during storage with Beyond extender.

Although the chemically defined extender promoted sperm metabolism, it did not confer good sperm membrane protection. The percentages of sperm with intact membrane and intact acrosome were lower with Beyond than with BotuSemen Special and INRA 96 extenders pre‐ and post‐storage at both temperatures. These observations suggest that extender formulations designed to better preserve sperm membranes (Aurich 2005; Becerro‐Rey et al. 2024; Umair et al. 2023) might improve the performance of long‐term extenders. Sperm chromatin structure analysis results should be interpreted with the caveat that samples were inadvertently thawed during transit to the laboratory performing the analysis and were re‐frozen prior to evaluation. In fact, COMPαt values at Day 0 seemed slightly greater (~20%) than the normal reference ejaculate used in the laboratory for quality control (~11%). Nevertheless, since all the samples were subjected to the same conditions, we decided to include the results in this manuscript. As previously reported for stallion semen (Love et al. 2002; Wach‐Gygax et al. 2017), sperm chromatin damage increased during storage. Detrimental changes seemed more pronounced with storage at 17°C, with significant changes already observed at 2 days of storage. Significant detrimental changes were observed only after 7 days of storage at 5°C. Although the extender did not affect sperm chromatin with storage at 17°C, INRA 96 yielded the lowest percentage of sperm DNA damage, while BotuSemen Special yielded the highest COMPαt values at 5°C. The effect of Beyond on sperm DNA damage during storage was comparable to milk and milk‐based extenders.

Total sperm used for insemination is also important and the AI dose used in the present study (mean 2.58 × 109 sperm) was greater than in most studies. However, total progressively motile sperm at the time of insemination (0.91 × 109 sperm) was similar to those reported in previous studies using sperm preserved in milk and milk‐based extenders at 5°C for 12–48 h (Douglas‐Hamilton et al. 1984; Kareskoski et al. 2019; Varner et al. 1989). In the only study reporting fertility of stallion sperm stored at 5°C for more than 48 h, pregnancy rates obtained after 70 or 80 h of storage were similar (65% overall). Total sperm number in the breeding dose in that study was 2 × 109 sperm with 0.57 × 109 progressively motile sperm at the time of insemination (Heiskanen et al. 1994). Since in the current study differences between fertile and non‐fertile AI doses were not observed for the number of motile, progressively motile, or progressively motile morphologically normal sperm, no inferences could be made about the effect of insemination dose after long‐term semen storage. Predicting sperm motility decline during storage might be useful for creating packaging instructions and calculating insemination doses according to the expected day of use. When sperm motility data for semen processed with Beyond extender in Experiments I and II were pooled, the relative (from the ejaculate) total and progressive sperm motility after storage at 5°C for 7 days were 63% and 45%, respectively (Figure S6).

The long‐term extender seemed to not only prolong sperm survival in vitro but also to promote in vivo longevity. Cooled semen insemination more than 24 h before ovulation has been associated with significantly reduced pregnancy rate (29%) when compared to inseminations 24 h before to 16 h after ovulation (60%–70%) (Newcombe and Cuervo‐Arango 2011). Similarly, positive effects of multiple inseminations during the same cycle on pregnancy rates have been attributed to the proximity to ovulation more than to total inseminated sperm number (Kareskoski et al. 2019; Squires et al. 1998). However, in the present study, fertility when ovulation occurred within 24 h after insemination (58%, 7/12) or within 24–48 h after insemination (67%, 4/6) did not seem to differ. Since examinations were only performed at 24 h intervals, the variation in the actual time of ovulation within groups was likely considerable. Nonetheless, these are important practical observations since being able to combine ovulation‐induction treatment with insemination might facilitate breeding management. Administration of ovulation‐inducing hormones such as deslorelin in mid oestrus reliably induces ovulation of dominant follicles 24–48 h after administration, and their use is recommended for the management of mares for AI (McCue 2021). Evaluation of long‐term extender exposure on more prolonged in vivo survival is warranted.

The ability to preserve cooled stallion semen longer than 72 h cannot be attributed only to the use of a long‐term extender. Successful long‐term storage required using an entire package of semen processing techniques, including the addition of antibiotics to the semen extender, seminal plasma removal and temperature control. These techniques have been shown to improve the quality and fertility of stallion cooled semen (Aurich 2008; Clulow and Gibb 2022; Loomis 2006; Varner 2016). Failure to properly process semen for long‐term storage might result in bacterial growth and reduced sperm longevity, ultimately resulting in poor fertility that might not be related to the quality of the long‐term extender itself.

Gross bacterial contamination as evidenced by observation of bacteria during sperm motility analysis or coagulation of the sample was observed by the end of storage at 17°C. Antibiotics are commonly added to equine semen extenders to control bacterial growth, preserve semen quality and avoid transmission of pathogenic organisms. The extender used for initial extension and centrifugation in the present study contained amikacin 1 mg/mL and penicillin 0.640 mg/mL. These antibiotics, used alone or in combination, have been shown to reduce bacterial growth in stallion cooled and frozen semen (Brito et al. 2023; Hernandez‐Aviles et al. 2020, 2018). Neat semen was incubated with extender containing antibiotics for 30 min prior to 20 min centrifugation. Incubation is important to ensure adequate exposure time to antibiotics. In Experiment II, neat semen was incubated and centrifuged with the extender with antibiotics prior to splitting the sperm pellets for final extension. Therefore, all groups were subjected to the same initial antibiotic treatment, but the final antibiotics concentration differed according to the final extender. INRA 96 contains gentamicin 0.105 mg/mL, penicillin 0.038 mg/mL and amphotericin B 0.315 mg/mL (IMV, personal communication). Beyond contains amikacin 1 mg/mL, penicillin 0.640 mg/mL and nystatin 0.0228 mg/mL. BotuSemen Special does not seem to contain antibiotics. Antibiotic additives to semen extenders and pre‐centrifugation incubation did not suffice to control bacterial growth as demonstrated by gross contamination of several samples stored at 17°C for 7 days. Although bacterial cultures were not performed, cooling seemed essential to control bacterial growth as no gross contamination was observed during storage at 5°C for 14 days. The different rates of bacterial growth at 17°C and at 5°C indicate that the antibiotics had a bacteriostatic rather than bactericidal effect.

Seminal plasma is not an ideal medium, and high concentrations are detrimental to semen stored in a liquid state. Semen extension rate > 1:2 or removal of seminal plasma by centrifugation has been reported to improve sperm motility and DNA integrity during cold storage (Brinsko et al. 2000; Jasko et al. 1991; Love et al. 2005; Varner et al. 1987). In the present study, the supernatant in the centrifuging tube containing the initial extender was removed almost entirely prior to re‐suspension of the sperm pellet into the desired final extender, and the remaining seminal plasma content was minimal. The effect of seminal plasma on long‐term sperm storage warrants study. Centrifugation is the most common method used to remove seminal plasma from stallion semen, but it must be performed with care to minimise sperm damage (Alvarenga et al. 2016; Loomis 2006). Sperm motility decreased after the required initial semen processing that involved incubation of extended semen at room temperature for 30 min followed by cushion centrifugation at 1000×g for 20 min. It is unclear which of these steps contributed to the observations; however, centrifugation (500×g for 18 min) has been shown to result in immediate sperm motility reduction, but the effect was no longer significant after 24 h of cold storage (Jasko et al. 1991). Evaluation of techniques to remove seminal plasma that reduces sperm damage during initial processing might improve results with long‐term storage extenders.

The optimal storage temperature for long‐term preservation of stallion liquid semen is approximately 5°C (Moran et al. 1992; Varner et al. 1988, 1989). Sperm tolerates rapid cooling from body temperature to 20°C, but cooling must be done slowly between 20°C and 5°C to prevent cold shock, a phenomenon associated with irreversible, deleterious changes to the sperm membranes (Amann and Pickett 1987; Moran et al. 1992). Although semen temperature was not evaluated in the present study, the cooling rate between 20°C and 5°C obtained with an Equitainer is −0.04°C to −0.05°C/min, which is within the optimal range for stallion sperm (Brinsko et al. 2000; Moran et al. 1992; Varner et al. 1988). Different cooling rates might adversely affect semen quality, especially if the cooling rate is too fast. For these reasons, semen samples were initially packaged into an Equitainer to provide an optimum, standardised cooling rate to 5°C. However, the Equitainer is a passive cooling device that cannot maintain constant cold temperature for much more than 48 h. Therefore, semen samples were removed from the Equitainer after 24 h of storage and transferred to a fridge for long‐term 5°C storage. Special attention must be paid to the cooling process and to maintaining stable cold temperature for proper long‐term semen preservation.

Semen storage at room temperature is usually reserved for semen used for insemination within 24 h after semen collection. In one study, pregnancy rates obtained with semen stored in INRA 96 for 24 h were similar for storage temperatures of 4°C and 15°C (Batellier et al. 2001). In this study, 17°C was used as that is the temperature recommended by Beyond's manufacturer. Sperm motility with Beyond was very similar throughout 1 week of storage regardless of storage temperature, and fertility evaluation of stallion semen after long‐term storage at 17°C is warranted. BotuSemen Special differed in its ability to support sperm motility depending on storage temperature. Although sperm motility with this milk extender supplemented with cholesterol followed closely that of milk‐based extender INRA 96 at 5°C, sperm motility decreased very sharply when BotuSemen Special samples were stored at 17°C. Sperm motility with INRA 96 was somewhat similar for 17°C and 5°C until approximately 3 days of storage, after which a sharper decline with the greater temperature was evident.

In conclusion, Beyond extender resulted in greater sperm motility longevity when compared to milk and milk‐based extenders, especially when semen was stored at 5°C. Satisfactory fertility was obtained when semen cooled and stored for 4–8 days was used for AI.

Author Contributions

Leonardo F. C. Brito: conceptualization, methodology, funding acquisition, project administration, investigation, data curation, formal analysis, writing – original draft. Renata L. Linardi: investigation, writing – review and editing. Leslie A. S. Rosales: investigation. Nithiya Sri Balamurugan: investigation. Camilo Hernández‐Avilés: investigation, writing – review and editing. Luisa Ramírez‐Agámez: investigation.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: rda70126‐sup‐0001‐AppendixS1.pdf.

Figure S2: rda70126‐sup‐0001‐AppendixS1.pdf.

Figure S3: rda70126‐sup‐0001‐AppendixS1.pdf.

Figure S4: rda70126‐sup‐0001‐AppendixS1.pdf.

Figure S5: rda70126‐sup‐0001‐AppendixS1.pdf.

Figure S6: rda70126‐sup‐0001‐AppendixS1.pdf.

Table S1: rda70126‐sup‐0001‐AppendixS1.pdf.

Table S2: rda70126‐sup‐0001‐AppendixS1.pdf.

Table S3: rda70126‐sup‐0001‐AppendixS1.pdf.

Table S4: rda70126‐sup‐0001‐AppendixS1.pdf.

Table S5: rda70126‐sup‐0001‐AppendixS1.pdf.

RDA-60-e70126-s001.pdf (697.2KB, pdf)

Acknowledgements

This study was supported by the Georgia and Philip Hofmann Endowment. The authors would like to thank Dr. Elizabeth Suarez for her assistance with the breeding trial and the staff from the Hofmann Center for Animal Reproduction, especially Walter Guessford and Jess Edstrom for their assistance with handling and caring for the horses used in this study. Drs. Hernández‐Avilés and Ramírez‐Agámez's research at Texas A&M University is funded by the Legends Premier Stallion Season Auction, the Department of Large Animal Clinical Sciences and AgriLife start‐up funds.

Brito, L. F. C. , Linardi R. L., Rosales L. A. S., Balamurugan N. S., Hernández‐Avilés C., and Ramírez‐Agámez L.. 2025. “Evaluation of a Chemically Defined, Long‐Term Extender for Liquid Storage of Stallion Semen.” Reproduction in Domestic Animals 60, no. 9: e70126. 10.1111/rda.70126.

Funding: This work was supported by the Georgia and Philip Hofmann Endowment and Legends Premier Stallion Season Auction.

Data Availability Statement

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

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: rda70126‐sup‐0001‐AppendixS1.pdf.

Figure S2: rda70126‐sup‐0001‐AppendixS1.pdf.

Figure S3: rda70126‐sup‐0001‐AppendixS1.pdf.

Figure S4: rda70126‐sup‐0001‐AppendixS1.pdf.

Figure S5: rda70126‐sup‐0001‐AppendixS1.pdf.

Figure S6: rda70126‐sup‐0001‐AppendixS1.pdf.

Table S1: rda70126‐sup‐0001‐AppendixS1.pdf.

Table S2: rda70126‐sup‐0001‐AppendixS1.pdf.

Table S3: rda70126‐sup‐0001‐AppendixS1.pdf.

Table S4: rda70126‐sup‐0001‐AppendixS1.pdf.

Table S5: rda70126‐sup‐0001‐AppendixS1.pdf.

RDA-60-e70126-s001.pdf (697.2KB, pdf)

Data Availability Statement

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

Articles from Reproduction in Domestic Animals = Zuchthygiene are provided here courtesy of Wiley

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