Effects of glucose concentration in semen extender and storage temperature on stallion sperm quality following long-term cooled storage

Theriogenology 147 (2020) 1e9

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Effects of glucose concentration in semen extender and storage temperature on stallion sperm quality following long-term cooled storage
ndez-Avile
s*, Charles C. Love, Rosanna Serafini, Luisa Ramírez-Aga
mez, Camilo Herna Macy Friedrich, Sharmila Ghosh, Sheila R. Teague, Katrina A. LaCaze, Steven P. Brinsko, Dickson D. Varner Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 500 Raymond Stotzer Parkway, College Station, TX, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 November 2019 Received in revised form 4 February 2020 Accepted 5 February 2020 Available online 11 February 2020

In Experiment 1, the effects of glucose concentration in extender (0 mM, 67 mM, 147 mM, 270 mM; G0, G67, G147, and G270, respectively) and storage temperature of extended semen (5, 10, 15 and 20
C) were evaluated after storage for up to 5 days (T0h to T120h). For all time points tested, mean total (TMOT) and progressive (PMOT) sperm motility were lower in G0 than all other treatment groups (P < 0.05). Mean curvilinear velocity (VCL) was lower in G0 than other treatment groups at all time points tested except T0h (P < 0.05). Mean percentage of plasma membrane/acrosome intact sperm (VAI) was similar among treatments at T0h, T72h, and T120h (P > 0.05). Mean TMOT and PMOT, were lower for semen stored at 20
C than all lower storage temperatures (P < 0.05) at all time points. In Experiment 2, semen was stored at 10
C in extender containing no added glucose (G0) or 147 mM glucose (G147). Following storage, semen was centrifuged and resuspended in extender containing no added glucose (G0 e G0 or G147 e G0, respectively) or 147 mM of glucose (G0 e G147 or G147 e G147, respectively). Mean TMOT, PMOT, and VCL were higher in G147 than G0 at all time periods tested (P < 0.05), whereas mean VAI was similar between these treatment groups throughout the experiment (P > 0.05). Mean TMOT and PMOT were higher in G0 e G147 than G0 e G0 at T72h and T120h (P < 0.05) and mean VCL was higher in G0 e G147 than G0 e G0 for all time periods. Mean TMOT, PMOT, and VCL were higher in G147 e G147 than G147 e G0 at all time points tested (P < 0.05), whereas mean VAI was similar between these two treatment groups for each of the time points (P > 0.05). In Experiment 3, the minimum concentration of glucose required to maintain sperm quality following long-term cooled storage (T120 h) was evaluated (G0, G5, G10, G20, G40, G67, G147 mM). At T120 h, mean TMOT was lowest in G0, G5, G10, and G20 (P < 0.05), whereas mean PMOT and VCL were lower in G0, G5, G10, and G20 than in G40, G67, and G147 (P < 0.05). Mean VAI was higher in G10 than G67, but similar among G10 and other treatment groups (P > 0.05). In conclusion, the absence of added glucose in extender reduced the motion characteristics of stallion sperm during long-term storage (5 days), but VAI was not affected. The use of temperatures between 5 and 15
C for long-term storage (5 days) best maintained sperm motility and VAI. The threshold concentration of added glucose in extender required to optimize sperm motion characteristics was 40 mM. Published by Elsevier Inc.

Keywords: Stallion sperm Glucose Temperature Motility Viability

1. Introduction Stallion semen is frequently subjected to cooled storage prior to

* Corresponding author.
s). E-mail address: [email protected] (C. Hern
andez-Avile https://doi.org/10.1016/j.theriogenology.2020.02.007 0093-691X/Published by Elsevier Inc.

insemination of mares; yet, this procedure is often associated with lower pregnancy rates, as compared to natural cover or artificial insemination using fresh semen [1e3]. Decreased sperm quality following cooled storage is thought to be a major contributing factor to reduced fertility [4,5], with associated decreases in sperm motility, plasma membrane intactness, and chromatin quality [6,7], and a corresponding increase in the reactive oxygen species (ROS)

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[8,9]. Commercially available equine semen extenders for cooled storage are formulated with concentrations of glucose that are much higher (67e270 mM [10,11]) than that present in the equine reproductive tract (seminal plasma - 0.38 to 2.7 mM [12,13]; oviductal fluid - 110 to 370 mM [14]). The importance of glucose as an energy substrate for equine sperm has been questioned, with recent research revealing that equine sperm rely primarily on the oxidative phosphorylation (OXPHOS) pathway to produce the ATP necessary for the maintenance of motility and plasma membrane intactness [15,16]. Furthermore, the metabolizable substrates, lactate or pyruvate (which are directly incorporated into the OXPHOS pathway) are associated with higher mitochondrial activity and reduced ROS production in stallion sperm, as compared to glucose [17]. It is unknown whether high glucose concentration in equine semen extender could alter sperm quality, or if its presence is necessary to optimize sperm quality following cooled storage. The ideal protocol for cooling and storing equine semen remains unknown. Chanavat et al. reported that a storage temperature of 15
C yielded lower sperm motility than 4
C; however, peroxidative injury, membrane phospholipid disorder, and calcium influx were lessened at the higher temperature [18]. Parks and Lynch found that the peak thermotropic phase transition temperature for phospholipids in equine sperm membranes was 20e21
C [19], and others have reported that equine sperm in milk-based extender could only tolerate rapid cooling to 19e20
C [20,21]. As such, some investigators are proposing ambient-temperature storage conditions for equine sperm to avoid the potentially detrimental effect of lower temperatures on sperm membrane composition [22,23]. Nonetheless, conflicting results regarding sperm quality or stallion fertility have been reported when storage temperatures between 4
C and 20
C have been compared [24e31]. Three experiments were conducted to examine: the effects of glucose concentration in extender and temperature of stored semen on sperm quality following a 5-day storage period; the ability of sperm resuspension in fresh extender containing either no-added or 147-mM glucose to recover sperm quality; the threshold glucose concentration required for maintenance of sperm quality following cooled storage. 2. Materials and methods 2.1. Media and reagents Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). The base semen extender consisted of nonfat dried milk solids (Sanalac®, ConAgra Foods, Inc., Omaha, NE, USA; 24 g/L, amikacin disulfate (1 g/L), and potassium penicillin G (1  106 IU/L), in deionized water. In Experiment 1, 4 different added glucose concentrations were examined: 0 mM (G0), 67 mM (G67), 147 mM (G147), and 270 mM (G270). A non-penetrating disaccharide, sucrose, was added to each glucose treatment to adjust extender osmolality to 350e360 mOsm/kg [61] as follows: G0 e 270 mM sucrose; G67 e 212 mM sucrose; G147 e 116 mM sucrose; G270 e no sucrose. Sodium bicarbonate was added to adjust extender pH to 6.8e6.9. The glucose concentrations selected represented those in some commercially available extenders: 67 mM glucose in INRA-96® extender (IMV International, L’Aigle, France; [32]); 147 mM glucose in Texas A&M University extender [33] or EquiPRO® (Minitube of America, Verona, WI, USA); and 270 mM glucose in Kenney-type Extenders [34] (EZ-Mixin® BF, ARS, Chino, CA, USA; Next Generation®, Exodus Breeders, York, PA, USA; BotuSemen®, Botupharma, Sao Paulo, Brazil). In Experiment 2, two glucose concentrations were tested:

0 mM glucose þ270 mM sucrose (G0) or 147 mM glucose þ 116 mM sucrose (G147). In Experiment 3, seven different glucose concentrations were tested: 1) 0 mM glucose þ270 mM sucrose (G0); 2) 5 mM glucose þ 242 mM sucrose (G5); 3) 10 mM glucose þ 231 mM sucrose (G10); 4) 20 mM glucose þ 219 mM sucrose (G20); 5) 40 mM glucose þ 207 mM sucrose (G40); 6) 67 mM glucose þ 199 mM sucrose (G67); or 7) 147 mM glucose þ 116 mM sucrose (G147). 2.2. Stallions and semen collection All animal procedures were carried out following Institutional Animal Ethics Committee guidelines. Stallions were light-breed, healthy, sexually active, and aged 5 to 23 y. In Experiment 1, one ejaculate from each of 7 stallions was used (n ¼ 7). In Experiment 2, three ejaculates from each of three stallions (aged 15 to 23 y) were used (n ¼ 9). In Experiment 3, three ejaculates from each of three stallions (aged 7 to 23 y) were used (n ¼ 9). As inclusion criteria, only stallions with initial sperm total motility and sperm plasma membrane intactness higher than 70% were used for all experiments. Prior to semen collection, each stallion was exposed to an ovariectomized mare to stimulate sexual arousal. Once erect, the penis was rinsed thoroughly with warm water and dried with paper towels. Ejaculates were collected using an artificial vagina (Missouri-model; Nasco, Ft. Atkinson, WI, USA) equipped with an in-line nylon micromesh filter (Animal Reproduction Systems, Chino, CA, USA) to permit collection of gel-free semen. Following semen collection, the gel-free semen was transported to an adjacent laboratory and placed in an incubator (37
C) prior to processing. 2.3. General semen processing The gel-free semen was weighed to determine volume, and sperm concentration was measured using a fluorescence-based cell counter (NucleoCounter SP-100 ™, Chemometec A/S, Allerød, Denmark) [35]. In Experiment 1, raw semen was divided into four aliquots and diluted in a 1:1 ratio (v/v) with extender at each of four different glucose concentrations (G0, G67, G147 or G270). In Experiment 2, raw semen was divided into two aliquots and diluted 1:1 (v/v) with extender at two different glucose concentrations (G0 or G147). In Experiment 3, raw semen was diluted 1:1 (v/v) with the extender at each one of the seven different glucose concentrations (G0, G5, G10, G20, G40, G67, G147). In all experiments, extended semen was subjected to cushioned centrifugation at 400g for 20 min using glass-nipple bottom tubes (Pesce Lab Sales, Kennett Square, PA, USA; [36]); Following removal of the supernatant, sperm pellets were resuspended to approximately 30  106 sperm/ mL using the same extender which contained 10% (v/v) fresh, homologous seminal plasma. Aliquots (1 mL) of extended semen were loaded into 1.2 mL plastic capped vials. 2.4. Experimental design and implementation 2.4.1. Experiment 1: The effect of glucose concentration and storage temperature on sperm motion characteristics and plasma membrane/acrosome intactness After centrifugation and resuspension, aliquots of extended semen containing each of four glucose concentrations (G0, G67, G147 or G270) were placed into active cooling devices (PC3 Controlled Cooler, Koolatron®, Brantford, ON, Canada), which were previously calibrated for four different temperatures to be tested (5, 10, 15 or 20
C), and stored up to 5 days. The mean (±SEM) temperatures inside the cooling devices were: 5.1 ± 0.3
C, 9.9 ± 0.7
C, 15.5 ± 0.8
C, and 20.5 ± 0.3
C. After 24, 72 and 120 h of storage


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(T24h, T72h, and T120h, respectively), aliquots of extended semen were evaluated for sperm motion characteristics and plasma membrane/acrosome integrity.

Experimental endpoints included: percentage of total motile sperm (TMOT); percentage of progressively motile sperm (PMOT) and mean curvilinear velocity (VCL; mm/s).

2.4.2. Experiment 2: The effect of post-storage sperm centrifugation/resuspension on sperm motion characteristics and plasma membrane/acrosome intactness Aliquots of extended semen containing two different glucose concentrations (G0 or G147) were stored at 10
C, using an active cooling device (PC3 Controlled Cooler) for up to T120 h. Mean (±SEM) temperature inside the cooling device was 10.1 ± 0.2
C. After T24h, T72h and T120h of storage, aliquots of extended semen were subjected to cushioned-centrifugation. Supernatant was removed and sperm pellets were resuspended in extender without (G0) or with (G147) added glucose. Briefly, extended semen was pipetted onto 1.5 mL tubes containing 30 mL of underlying cushion fluid (MaxiFreeze™, IMV International, L’Aigle, France). The tubes were centrifuged at 400g x 5 min in a fixed-angle rotor microcentrifuge (Eppendorf 5415D, Hamburg, Germany). Following centrifugation, supernatant was gently aspirated using a vacuum pump, and sperm pellets were resuspended to 1 mL using fresh extender with or without 147 mM glucose and containing 10% (v/v) homologous frozen seminal plasma from the same ejaculate collected at the beginning of each experiment. Six treatment groups were compared: 1) semen stored with 0 mM glucose and not centrifuged after storage (G0); 2) semen stored with 0 mM glucose, centrifuged and resuspended in 0 mM glucose extender after storage (G0 e G0); 3) semen stored with 0 mM glucose, centrifuged and resuspended in 147 mM glucose extender after storage (G0 e G147); 4) semen stored with 147 mM glucose and not centrifuged after storage (G147); 5) semen stored with 147 mM glucose, then centrifuged and resuspended in 0 mM glucose extender (G147 e G0); or 6) semen stored with 147 mM glucose, then centrifuged and resuspended in 147 mM glucose extender (G147 e G147). Following centrifugation and resuspension, semen aliquots were incubated at 37
C for 15 min, and then evaluated for sperm motion characteristics and plasma membrane/acrosome integrity.

2.6. Sperm plasma membrane/acrosome integrity

2.4.3. Experiment 3: The minimum glucose concentration in extender required to maintain sperm motion characteristics and plasma membrane/acrosome integrity Aliquots of extended semen containing seven different glucose concentrations (G0, G5, G10, G20, G40, G67 or G147) were stored at 10
C, using an active cooling device (PC3 Controlled Cooler) for up T120 h. The mean (±SEM) temperature inside the cooling device was: 10.1 ± 0.2
C. At T24h, T72h, and T120h, aliquots of extended semen were incubated at 37
C for 15 min and then evaluated for sperm motion characteristics and plasma membrane/acrosome integrity. 2.5. Sperm motion characteristics Sperm motion characteristics were analyzed by computerassisted sperm motion analysis (CASMA, IVOS II, HamiltonThorne, South Hamilton, MA, USA), as previously described [36]. A minimum of 10 fields and 500 sperm were measured for each sample. Pre-set values for the IVOS II system consisted of the following: frames acquired-45/s; frame rate-60 Hz; minimum contrast-70; minimum cell size-4 pixels; minimum static contrast30; straightness (STR) threshold for progressive motility-50%; average path velocity (VAP) threshold for progressive motility30%; VAP threshold for static cells-15 mm/s; cell intensity-106 pixels; static head size-0.60 to 2.00 mm; static head intensity-0.20 to 2.01; static elongation-40 to 85; illumination intensity-2200.

The integrity (intactness) of the sperm plasma membrane and acrosome membrane was simultaneously evaluated, as previously described [37]. An aliquot (40 mL) of extended semen (30  106 sperm/mL) was added to 133 mL of Dulbecco’s phosphate-buffered saline (DPBS; Invitrogen Gibco®, Carlsbad, CA, USA). Two mL of propidium iodide (Invitrogen Molecular Probes, Eugene, OR; 1.3 mM final concentration) and 10 mL Pisum sativum agglutinin (PSA)-FITC conjugate (Sigma-Aldrich; 0.05 mg/mL final concentration) were added to each sample. The samples were incubated at room temperature (approximately at 25
C) in the dark for 10 min. Then, 20 mL of this mixture were diluted with 400 mL Dulbecco’s Phosphate Buffered Solution (DPBS) and processed immediately by flow cytometry (FACScan, Becton Dickinson, Mountain View, CA, USA), equipped with a 488 nm argon laser. The voltage settings on the flow cytometer were: SSC 240, FL1 690 and FL2 657. The compensation was set at FL1 as 1.9% of FL2, and at FL2 as 18.8% of FL1. A minimum of 5000 events were evaluated per sample. Data were acquired using a log scale and analyzed by WinList™ software (Verity Software House, Topsham, ME, USA). The experimental endpoint was the percentage of the sperm population that contained intact plasma and acrosomal membranes (VAI). 2.7. Statistical analysis Statistical analysis was performed using proprietary software (SAS Version 9.4; SAS Institute, Inc., Corp., Cary, NC, USA). In all experiments, the Shapiro-Wilk test was conducted to test data distribution. As the data were not normally distributed, a ranktransformation procedure (PROC RANK) was performed before analysis using the general linear model (PROC GLM) procedure. All statistics were performed within each time period tested. Tabular data are presented as non-transformed values for ease of interpretation. The LS Means-Tukey test was used to separate main-effect means when treatment F ratios were significant (P < 0.05). 3. Results

Experiment 1. The effect of glucose concentration and storage temperature on sperm motion characteristics and plasma membrane/acrosome intactness No interactions between glucose concentration and storage temperature were observed at any time period studied (P > 0.05). The main effects of extender glucose concentration (G0, G67, G147 or G270) are presented in Fig. 1. For all time points tested (T0h to T120h), mean TMOT and PMOT were lower in Group G0 than all other treatment groups (P < 0.05), and these measures were also were lower in Group G67 than Group G270 at T24h and T120h (P < 0.05). Mean VCL was lower in Group G0 than other treatment groups at all time points tested except T0h (P < 0.05). Mean VAI was similar across treatments T0h, T72h, and T120h (P > 0.05), but was slightly higher in Group G67 than Group G270 at T24h (P < 0.05). The main effects of storage temperature (5, 10, 15, and 20
C) are presented in Fig. 2. Mean TMOT and PMOT, were lower for semen stored at 20
C than all lower storage temperatures (P < 0.05); yet, values were similar amongst storage temperatures of 5e15
C

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Fig. 1. The effect of glucose concentration (G0, G67, G147, or G270) on stallion sperm motion characteristics (A. total motility, B. progressive motility, C. curvilinear velocity, D. plasma membrane/acrosome intactness) after cooled storage for 24, 72 and 120 h (n ¼ 7 ejaculates). a, b, c Within time periods (T0h, T24h, T72h, T120h), different superscripts indicate significant differences at P < 0.05. Error bars represent SEM. G0: Semen stored in extender containing 0 mM added glucose. G67: Semen stored in extender containing 67 mM added glucose. G147: Semen stored in extender containing 147 mM added glucose. G270: Semen stored in extender containing 270 mM added glucose.

(P > 0.05). For all time points tested, mean VCL was lower for semen stored at temperature of 20
C than 10
C or 5
C (P < 0.05), whereas mean VCL was lower at a storage temperature of 15
C than 10
C or 5
C only at T120h (P < 0.05). Mean VAI was similar across storage temperatures at T24h (P > 0.05), but lower for a storage temperature of 20
C than all lower storage temperatures at both T72h and T120h (P < 0.05). Experiment 2. The effect of post-storage sperm centrifugation/ resuspension on sperm motion characteristics and plasma membrane/acrosome intactness The effect of extender type and post-storage centrifugationresuspension method on four experimental endpoints are presented in Fig. 3. Mean TMOT, PMOT, and VCL were higher in Group G147 than Group G0 at all time periods tested (P < 0.05), whereas mean VAI was similar between these treatment groups throughout the experiment (P > 0.05). Mean TMOT and PMOT were higher in Group G0 e G147 than G0 e G0 at T72h and T120h (P < 0.05) and mean VCL was higher in G0 e G147 than G0 e G0 for all time periods. Mean VAI, however, was lower in G0 e G147 than G0 e G0 at both T24h and T72h (P < 0.05), but not at T120h (P > 0.05). Mean TMOT, PMOT, and VCL were higher in G147 e G147 than G147 e G0 at all time points tested (P < 0.05), whereas mean VAI was similar between these two treatment groups for each of the time points (P > 0.05). Mean TMOT and PMOT were higher in G147 e G147 than

G0 e G0 at T72h and T120h (P < 0.05), and mean VCL was higher in G147 e G147 than G0 e G0 at all time points (P < 0.05). Mean VAI was similar between these two treatment groups for all time periods (P > 0.05). For all endpoints and times examined, no treatments yielded higher values than Group G147. Experiment 3. The minimum glucose concentration in the extender required to maintain sperm motion characteristics and plasma membrane/acrosome intactness Data are presented in Fig. 4. At T0h, mean TMOT was higher in Group G67 than Groups G5 and G147 (P < 0.05), whereas mean PMOT, VCL and VAI were similar across all treatments (P > 0.05). At T24h, mean TMOT and PMOT were lower in Groups G0 and G5 than Groups G40, G67, and G147 (P < 0.05), whereas mean VCL was lower in Group G0 than Group G67 (P < 0.05). Mean VAI was lower in Group G147 than Groups G10 and G40 (P < 0.05). At T72h, mean TMOT, PMOT, and VCL were highest in Groups G40, G67, and G147 (P < 0.05), Mean VAI was higher in Group G20 than Groups G0 or G67 (P < 0.05), and lowest in Group G147 (P < 0.05). By T120 h, mean TMOT was lowest in Groups G0, G5, G10, and G20 (P < 0.05), whereas mean PMOT and VCL were lower in Groups G0, G5, G10, and G20 than in Groups G40, G67, and G147 (P < 0.05). At this time period mean VAI was higher in Group G10 than Group G67, but similar among Group G10 and other treatment groups (P > 0.05).


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Fig. 2. The effect of storage temperature (5
C, 10
C, 15
C or 20
C) on stallion sperm motion characteristics (A. total motility, B. progressive motility, C. curvilinear velocity, D. plasma membrane/acrosome intactness) after cooled storage for 24, 72 and 120 h (n ¼ 7 ejaculates). a, b Within time periods (T0h, T24h, T72h, T120h), different superscripts indicate significant differences at P < 0.05. Error bars represent SEM.

4. Discussion The best approach for prolonging sperm function while in a cooled state remains unclear [reviewed by Refs. [23,38,39]]. Variability in sperm motility amongst different stallions after cooled storage of extended semen has been reported [6,40], but the percentage of stallions possessing semen with greatly reduced sperm function following cooled storage has not been reported. Such disruption in sperm function has been attributed to “cold-shock” induced by fast cooling rates between 19 and 8
C [21]. Incorporation of cholesterol-loaded cyclodextrin into extenders is reported to improve sperm function following cooled storage of semen from such stallions [40], as is pre-cooling centrifugation of semen to reduce the seminal plasma contribution in extended semen [6]. Given the fact that sperm from some stallions appear to be more sensitive to thermal effects, the use of temperatures higher than the “critical” range for stallion sperm (19e8
C) has been proposed as a method to maintain higher sperm quality [22]. The results observed in the present study suggest that temperatures above 15
C are deleterious to sperm motility after 24 h of storage, and sperm plasma membrane/acrosomal membrane intactness by 72 h of semen storage. These results might be explained by inadequate reduction in the metabolic rate of sperm during storage at higher temperatures coincident with accumulation and liberation of toxic by-products such as reactive oxygen species (ROS) [41]. Stallion sperm reportedly utilize the oxidative phosphorylation (OXPHOS) pathway to generate ATP for motility [15]. Higher storage

temperatures would result in intensified mitochondrial activity, with an associated increase in endogenous ROS generation and resultant membrane lipid peroxidation, as well as accelerated depletion of ATP and cell death [41e43]. Somatic cells with high metabolic capacity that rely on OXPHOS for energy production (i.e. neurons, hepatocytes) have enzymatic and metabolic rates reduced by approximately half for each 10
C reduction in temperature below body temperature [44,45]. If the same kinetics hold true for stallion sperm, this would mean that after stallion sperm is cooled to 18e20
C, metabolic activity would be approximately 25% of that at body temperature. Storage temperatures of 4e5
C are theorized to decrease sperm metabolism by 93% [46]. Others have suggested that a storage temperature of 4
C is more likely than 15
C to induce sperm lipid peroxidation, phospholipid disorder, and calcium influx; however, pregnancy rates were not different between the two groups following 3 days of storage [18]. Certainly, a wide range of temperatures between 4
C and 15
C have yielded similar pregnancy rates following cooled storage of stallion semen [30]. In Experiment 1, absence of added glucose in a milk-based extender had no detrimental effect on plasma/acrosomal membrane intactness of stallion sperm, but its presence was related to higher values for sperm motility characteristics after one to five days of semen storage. Others have observed that metabolic substrates which are readily incorporated into the OXPHOS pathway, such as pyruvate or lactate, could be more effective than glucose at maintaining motility values when semen is incubated for 1 h at 37
C [17] or maintained at room temperature for 72 h [47]. In

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Fig. 3. The effect of sperm centrifugation/resuspension using an extender with or without added glucose on stallion sperm motion characteristics (A. total motility, B. progressive motility, C. curvilinear velocity, D. plasma membrane/acrosome intactness) after cooled storage for 24, 72 and 120 h (n ¼ 9 ejaculates). a, b, c, d Within time periods (T0h, T24h, T72h, T120h), different superscripts indicate significant differences at P < 0.05. Error bars represent SEM. G0: Semen stored in extender containing 0 mM added glucose. G0 e G0: Semen stored in extender containing 0 mM added glucose, and then resuspended with extender containing 0 mM added glucose following storage. G0 e G147: Semen stored in extender containing 0 mM added glucose, and the resuspended with extender containing 147 mM glucose following storage. G147: Semen stored in extender containing 147 mM glucose. G147 e G0: Semen stored in extender containing 147 mM glucose, and then resuspended with extender containing 0 mM added glucose following storage. G147 e G147: Semen stored in extender containing 147 mM glucose, and the resuspended with extender containing 147 mM glucose following storage.

either of these studies, addition of glucose as a metabolic substrate had no effect on the percentage of sperm with intact plasma membranes [17,47], which agrees with our results. Others have also reported that elimination of glucose as a substrate did not impair plasma membrane integrity following storage for 4 h at 37
C [48]. Reduction of glucose concentration in extender also did not alter post-thaw membrane viability of cryopreserved sperm [49]. Collectively, these results suggest the maintenance of stallion sperm plasma membrane intactness is not related to the type or presence of supplemented metabolic substrate in extender. Possibly, simple inclusion of lipids and/or protein components in milk extender sufficiently stabilize the sperm plasma membrane and are protective against loss of integrity [50]. In Experiment 2, resuspension of stored semen (up to 5 days) with fresh extender containing glucose (147 mM) reactivated the motility of sperm previously stored in media without added glucose (0 mM). Following 5 days of cooled storage, sperm plasma/ acrosomal intactness was similar between the two groups. Conversely, resuspension of semen in fresh extender containing glucose did not improve features of sperm quality of semen previously stored in glucose-containing extender, as compared to semen that was stored in glucose-containing extender and not subjected to centrifugation/resuspension procedures. Although we

found a reduction in the percentage of plasma membrane/acrosome intact sperm after centrifugation/resuspension with fresh extender, as compared to the uncentrifuged samples, this reduction may be attributable to centrifugation technique (low volume centrifuge tubes in a fixed-rotor centrifuge). Previous studies from our laboratory revealed that resuspension of stallion sperm with fresh extender after cooled storage improved sperm motility without affecting sperm viability when semen was stored for up to 96 h [51]. Overall, no centrifugation/resuspension procedure increased measures of sperm quality following cooled storage above that observed in semen subjected to simple dilution in glucose-containing extender. Sperm plasma membrane/acrosomal intactness was less affected than sperm motility in this study. Previously, mares bred with cooled sperm (96 h) that were largely immotile but highly viable became pregnant at the same proportion as mares bred with highly motile and viable sperm [52]. The clinical relevance of sperm motility and viability requires further evaluation. In our study, glucose was selected as an energy substrate in the extender because it is the most common energy source included in commercial extender formulations for stallion semen. Incorporation of glucose in extender might be advantageous for long-term cooled storage of stallion sperm because sperm metabolism


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Fig. 4. The effect of glucose concentration (G0, G5, G10, G20, G40, G67 or G147) in extender on stallion sperm motion characteristics (A. Total motility, B. Progressive motility, C. Curvilinear velocity, D. Plasma membrane/acrosome intactness) after cooled storage for 24, 72 and 120 h (n ¼ 9 ejaculates). a, b, c, d Within time periods (T0h, T24h, T72h, T120h), different superscripts indicate significant differences at P < 0.05. Error bars represent SEM. G0 e G147: Semen stored in extender containing 0, 5, 10, 20, 40, 67- or 147-mM added glucose.

would be diverted initially from OXPHOS to glycolysis, possibly reducing the amount of ROS produced as a consequence of normal mitochondrial energy production [53]. Darr et al. demonstrated that use of glucose as a sole energy source in media held at 37
C led to a slight reduction of mitochondrial function as compared to pyruvate or lactate as energy substrates [17]. These workers proposed that stallion sperm are metabolically flexible, and capable of using the metabolic substrates available in the media to maintain energetic requirements. While glucose is not directly available for entry into the OXPHOS pathway, its simple presence in extender could be used for the preservation of other sperm-survival related functions, such as Na/K/ATPase pumps through passive glucose transporters in the principal piece [54]. Interestingly, studies conducted in other species whose sperm are thought to rely strictly on glycolysis for maintenance of motility (i.e. rats), have demonstrated that immotile sperm recovered from the cauda epididymis express high mitochondrial activity, whereas there is a shift towards glycolysis to maintain sperm motion characteristics following ejaculation [55]. Swegen et al. used the antidiabetic compound, rosiglitazone, to successfully divert sperm metabolism from OXPHOS toward glycolysis, thereby demonstrating the metabolic flexibility of stallion sperm [56]. The absence of additional glucose to extender yielded similar values of plasma membrane/acrosome intactness after long-term storage (5 days) in the current study, even though sperm motion characteristics were reduced. Experiment 3 was conducted to

ascertain the minimal glucose concentration required to maintain sperm motility following cooled storage. Based on the glucose concentrations detected in seminal plasma of stallions (0.38e2.7 mM [12,13]), we hypothesized that reducing the glucose concentrations in the extender to more “physiological levels” would still support sperm motility and plasma/acrosomal membrane intactness following cooled storage. We found no difference in sperm motion characteristics or plasma/acrosomal membrane intactness at any time point between extender containing no added glucose or extender containing 5 mM added glucose. The base milk extender was not glucose-free, as it was determined to contain approximately 1.1 mM glucose. Certainly, providing an added glucose concentration 40 mM generally enhanced sperm motion characteristics during cooled storage, as compared to added glucose concentrations 20 mM. A study conducted by Vasconcelos et al. [57] suggested that a glucose concentration approximately 6 mM maximized lactate and energy production (measured by calorimetry) while supporting sperm motility and plasma membrane function. The discrepancies between their study and the present report may be related to longer times of incubation (T2h vs T120h), and the presence of high quantities of seminal plasma in their study. Based on the results from the present study, we propose that reducing glucose concentration in milk-based extender to 40 mM should be considered because no advantages on sperm motion characteristics nor plasma membrane/acrosome intactness were observed at higher glucose concentrations.


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Glucose consumption rate by stallion sperm during in vitro conditions has not been reported. In bulls and rams, 2 mg fructose/ h/109 sperm (i.e., 0.01104 mmol/h) was required to maintain sperm motility at 37
C [58]. Stallion sperm do not metabolize fructose as efficiently as glucose [59], and stallion seminal plasma contains only scant amounts of fructose [58e60]. If glucose is metabolized in stallion sperm as efficiently as fructose is metabolized in bull and ram sperm, then 1  109 stallion sperm might consume 1.3248 mmol/5 d glucose when incubated at 37
C. If the sperm metabolic rate is reduced by 50% for every 10-degree reduction in storage temperature [46], glucose consumption for 1  109 sperm might be 0.185 mmol/5 d at 10
C. Our study evaluated 1 mL aliquots of extended semen containing 30  106 sperm, which would potentially utilize 0.0055 mmol glucose/5 d at 10
C. In Experiment 3, conducted at 10
C, the lowest glucose concentrations (i.e. G0 and G5; 0.0011 and 0.005 mmol/mL, respectively) yielded the lowest motility values among all treatments. This might be explained by utilization of all glucose for maintenance of sperm motility, given the theoretical amounts of glucose utilization mentioned above. However, this does not explain why sperm motility was also lower for semen stored in G10 or G20, as compared to G40, G67 or G147. This suggests that the calculated values based on bull and ram data [58] may be low for stallion sperm. Therefore, further studies are needed to examine glucose metabolism of stallion sperm during cooled storage. In conclusion, the absence of added glucose in the extender reduced motion characteristics of stallion sperm during long-term storage (5 days), while sperm plasma membrane/acrosome intactness was unaffected. Storage temperatures of 5, 10, and 15
C yielded higher values than 20
C for maintaining both sperm motility and plasma membrane/acrosome intactness. Reducing glucose concentration in extender to 40 mM effectively maintained both sperm motility and plasma membrane/acrosomal intactness following cooled storage for up to 5 days. Authors’ contributions CH-A, RS, LR-A, MF, SG, SRT and KALaC contributed to experimental procedures and data acquisition. CH-A, CCL, SPB and DDV contributed to experimental design, statistical analysis and manuscript preparation Source of funding The study was funded by the Legends Premier Stallion Season Auction Fund, Texas A&M University. Declarations of competing interest None. References [1] Squires E, Barbacini S, Matthews P, Byers W, Schwenzer K, et al. Retrospective study of factors affecting fertility of fresh, cooled, and frozen semen. Equine Vet Educ 2006;18:96e9. [2] Jasko DJ, Moran DM, Farlin ME, Squires EL, Amann RP, Pickett BW. Pregnancy rates utilizing fresh, cooled and frozen-thawed semen. In: Proc 38th Ann conv amer assoc equine pract; 1992. p. 649e60. [3] Brinkerhoff JM, Love CC, Thompson JA, Blodgett G, Teague SR, Varner DD. Influence of mare age, pre-breeding status, breeding method, and stallion on first cycle pregnancy rates on a large commercial breeding farm. Anim Reprod Sci 2010;121S:S159. [4] Heckenbichler S, Deischel K, Peters P, Aurich C. Quality and fertility of cooledshipped stallion semen at the time of insemination. Theriogenology 2011;75(5):849e56. [5] Love CC, Noble JK, Standridge SA, Bearden CT, Blanchard TL, et al. The relationship between sperm quality in cool-shipped semen and embryo recovery

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