Clinical Theriogenology 2022; 14: 356 Sucrose and bovine serum albumin in association with dimethylformamide improved sperm parameters of frozen stallion semen Madeline Kurpita,a Jose Lena,b aUniversity of Adelaide School of Animal and Veterinary Sciences, Australia bNorth Carolina State University College of Veterinary Medicine, Raleigh, NC Abstract Postthaw stallion sperm integrity was determined after freezing semen in an extender supplemented with several sucrose concen- trations, bovine serum albumin (BSA), and with and without dimethylformamide (DMF). Two ejaculates from 6 stallions (n =12) were diluted and aliquots (n = 7) were made for treatment groups (25 mM sucrose + BSA [S25], S25 + DMF [S25DMF], 50 mM sucrose + BSA [S50], S50 + DMF [S50DMF], 100 mM sucrose + BSA [S100], S100 + DMF [S100DMF], and Control (only DMF). Se- men was frozen in a computer-controlled freezer. Sperm postthaw motility (total and progressive) and kinetics were assessed using CASA. Postthaw sperm plasma membrane and acrosomal integrity were evaluated using SYBR-14/PI and FITC-PNA, respectively. Sperm motility was higher in S100DMF and S50DMF. Plasma membrane integrity was higher in S100DMF, S50DMF, and S100. As sucrose concentration increased, plasma membrane integrity increased. Treatment groups with sucrose and BSA, regardless of DMF, had higher acrosome integrity than Control. Sucrose and BSA in association with DMF in a freezing extender protected sperm integrity during freezing and thawing. Keywords: Stallion sperm, freezing, sucrose, dimethylformamide, plasma membrane Introduction Successful semen cryopreservation enhances benefits (long-dis- tance semen transport, international trade, and extended use of superior genetic material) of artificial insemination (AI) over natural breeding.1,2 Glycerol (GLY) is commonly used as a permeable cryoprotectant for stallion semen cryopreservation; however, GLY reduced sperm fertility.3 Dimethylformamide (DMF) was as effective as GLY in protecting stallion sperm from cryodamage.4,5 Furthermore, DMF improved postthaw sperm motility and plasma membrane integrity. Nonpermeable cryoprotectants (e.g., sucrose and trehalose) had beneficial effects during freezing in several species (rodents,7,8 rabbits,9 cattle,10 sheep,11,12 and pigs13). In stallions, sucrose14-16 and trehalose16,17 were used. A skim-milk egg yolk (SMEY) ex- tender with sucrose and bovine serum albumin (BSA) instead of GLY14 improved stallion sperm postthaw sperm motility and plasma membrane integrity compared to sperm frozen in a SMEY-GLY freezing extender. Furthermore, donkey semen fro- zen in a SMEY freezing extender with sucrose and BSA without GLY had similar sperm postthaw motility and higher plasma membrane integrity compared to semen frozen with a SMEY- GLY freezing extender.15 Improved sperm postthaw motility and plasma membrane were attributed to sucrose effects in re- ducing osmotic stress9 and BSA preventing lipid peroxidation.18 Cryopreservation of stallion semen with sucrose and BSA in combination with DMF has not been reported. We investigated sperm postthaw integrity of semen frozen: a) in extenders con- taining DMF, several sucrose concentrations, and 1% bovine serum albumin (BSA); b) in extenders without DMF, several sucrose concentrations, and 1% bovine serum albumin (BSA); and c) extender with only DMF. Materials and methods Animals During summer in the Southern Hemisphere, semen was col- lected from stallions (n = 6) of known fertility, ranging in age from 3 to 16 (10.8 ± 3.5) years. Except for 1 stallion (semen was frozen earlier using DMF) semen freezing capability was not known. Animal use and handling procedures were approved (University of Adelaide Office of Research Ethics, Compliance, and Integrity). Semen collection Stallions that were not actively breeding had 3 semen collec- tions prior to use.19 Two days after the last semen collection, stallions had their semen collected for freezing, a day apart be- tween the first and second semen collection. Semen was collect- ed using a Hannover or Missouri-model artificial vagina with an inline gel filter (Minitube Australia, Australia). Stallion had Clinical Theriogenology 2022; 14: 357 their penis washed with warm water and collection was per- formed over a phantom mount. Semen preparation Immediately after collection, total volume of gel-free semen was determined using a 50 ml conical tube (Corning®, MA), and sperm concentration (106/ml) was determined using an automated cell counter (NucleoCounter SP-100, ChemoMe- tec, Denmark). Sperm motility and membrane integrity were assessed (described later). Semen from ejaculates (n = 12) was extended 1:1 with Equiplus (Minutube Australia) and centri- fuged for 10 minutes at 1,000 x g. Supernatant was aspirated with a vacuum, sperm pellet was resuspended and diluted with Equiplus to a concentration of 400 x 106 sperm/ml. Resuspend- ed semen was transferred to semen freezing room (18°C) and divided into 7 equal aliquots. Experimental design Treatment groups (Table 1) were: S25 (Equiplus, 1% [w/v] BSA and 25 mM of sucrose [Sigma-Aldrich, Australia]); S25DMF (S25 with 2% DMF); S50 (Equiplus, 1% BSA and 50 mM of su- crose); S50DMF (S50 with 2% DMF); S100 (Equiplus, 1% BSA and 100 mM of sucrose); S100DMF (S100 with 2% DMF); and Control (Equiplus with 2% (v/v) DMF (Sigma-Aldrich). Freez- ing extenders were prepared in advance, divided into aliquots and stored at – 80°C until used. Because semen samples were diluted (1:1) with treatment groups before freezing, freezing extenders were prepared at double the cryoprotectants concen- tration to reach the final concentration (200 x 106 sperm/ml) of treatment groups after dilution. After dilution, samples’ osmo- lality was assessed using an osmometer (AdvancedTM Micro-Os- mometer, Model 3 MO Plus, ThermoFisher Scientific, Waltham, MA) that ranged from 379 to 769 mOsm kg-1 (Table 1). Table 1. Freezing extenders and osmolality (mOsm kg-1) of each extender Treatment Freezing extender mOsm kg-1 Control Equiplus + DMF 635 S25 Equiplus + sucrose (25 mM) + BSA 379 S25DMF Equiplus + sucrose (25 mM) + BSA + DMF 556 S50 Equiplus + sucrose (50 mM) + BSA 401 S50DMF Equiplus + sucrose (50 mM) + BSA + DMF 657 S100 Equiplus + sucrose (100 mM) + BSA 447 S100DMF Equiplus + sucrose (100 mM) + BSA + DMF 769 DMF = 2% v/v (0.26 M) dimethylformamide BSA = 1% w/v bovine serum albumin Semen freezing Semen was frozen using a computer-controlled rate freezer (IceCube 14 S-A 230V, Minitube Australia). Semen samples were then drawn into 0.5 ml plastic straws (Minitube Australia) and sealed with a sealing ball. Straws were placed horizontal- ly into the computer-controlled rate freezer. Cooling rate from 18°C to 4°C was 0.3°C/minute and from 4°C to –160°C was 60°C/minute. Straws were then plunged into liquid nitrogen (–196°C) and stored in liquid nitrogen tanks. After at least a week after freezing, 2 straws from each treatment group were thawed in a water bath at 37°C for 30 seconds. Sperm motility After collection, an aliquot (1 ml) of fresh semen was extend- ed with warm (37°C) Equiplus to a concentration of 50 x 106 sperm/ml before motility assessment. Postthaw sperm motility was assessed immediately after thawing at (0,15, and 30) min- utes. Before sperm motility assessment of frozen-thawed semen and to maintain osmolality consistency among treatments, each sample was diluted with its corresponding treatment to a con- centration of 50 x 106 sperm/ml. Five μl of fresh and/or fro- zen-thawed semen were placed over a warm glass slide (25 x 75 mm), covered with a coverslip (22 x 22 mm) and 3 fields were evaluated at 200 x magnification using CASA (AndroVision®, Minitube Australia). Analysis was performed using an image capture of 60 frames/second. Sperm motility and kinematics assessed were: total motility (TM %, VCL < 35 μm/s and VSL < 15 μm/s), progressive motility (PM %, VCL ≥ 35 μm/s and VSL ≥ 15 μm/s), sperm curvilinear velocity (VCL, μm/s), sperm linear velocity (VSL, μm/s), average path velocity (VAP, μm/s), amplitude of lateral head displacement (ALH, μm), and beat- cross frequency (BCF, Hz/s). Plasma and acrosomal membrane integrity Fresh and thawed semen were diluted to a 1 x 106 sperm/ml with phosphate buffered saline (osmolality adjusted to match treat- ment groups). For sperm plasma membrane integrity (PMI), diluted semen was mixed with 1 μl of the SYBR-14 and propid- ium iodide (SYBR-14/PI) solution (Reference [15407/0001], Minitube Australia) and incubated in dark for 10 minutes. Flu- orescent probes were excited with 488 nm (SYBR-14) and 561 nm (PI) lasers. Sperm emitting in red wavelength were consid- Clinical Theriogenology 2022; 14: 358 ered plasma membrane ‘damaged’ and sperm emitting in green wavelength were considered ‘intact’. For acrosome membrane integrity (ACR), an aliquot of semen was diluted to a 1 x 106 sperm/ml, mixed with 1 μl of the Fluo- rescein isothiocyanate (FITC) – peanut agglutinin (PNA) stain (Sigma-Aldrich) and incubated in the dark for 10 minutes. Flu- orescent probe was excited with a 488 nm laser. Sperm emitting in green wavelength were considered ‘damaged’ and unstained sperm were considered ‘intact’. For plasma and acrosomal membrane, assessment was made in duplicates from each sample using an Attune NxT Flow Cy- tometer (Thermofisher, Australia). At least 10,000 sperm were evaluated before and after cryopreservation. Data analyses Equality of variance was assessed using Levene’s test. Square root of VCL (0 and 30 minutes) and ALH (0 minute) were used for analysis. Effect of treatment on the mean (± SEM) postthaw TM (%), PM (%), VCL, VSL, VAP, ALH, and BCF at 0, 15, and 30 minutes were evaluated using analysis of variance (ANOVA; IBM SPSS statistics 26, US). Effect of treatment on the mean (± SEM) percentage postthaw PMI and ACR was also assessed using ANOVA. Level of significance was set at p < 0.05. When significance was observed, a pairwise comparison using Tukey’s method was performed to assess differences among treatments. Results Ejaculates (n = 12) had the following (mean ± SEM) character- istics before freezing: gel-free volume = 58.9 ± 7.8 ml, concen- tration = 205.1 ± 30.3 x 106/ml, TM = 73.4 ± 2.3%, PM = 72.2 ± 2.3%, intact PMI = 71.1 ± 2.3%, and intact ACR = 91.3 ± 0.7%. There was no difference (p < 0.05) among ejaculates for the parameters evaluated. There was an effect (p < 0.05) of treatment on postthaw sperm motility and kinematic parameters. Postthaw sperm motility (TM and PM) and sperm kinematic parameters (VCL, VSL, VAP, BCF, and ALH) at 0, 15, and 30 minutes in treatment groups S50DMF, S100DMF, and Control were the highest (Table 2 and 3). Stallion sperm frozen in treatment groups S25 and S100 had the lowest postthaw sperm motility (TM and PM) and kinemat- ics in most of the measured time points (Figures 1 and 2). Table 2. Postthaw (mean ± SEM) TM and PM at 0, 15, and 30 minutes of stallion sperm frozen with different sucrose concentra- tions +/- DMF (2%) and BSA (1%) Treatment Time (minutes) TM (%) PM (%) DMF 0 32.1 ± 4.3ab 31.0 ± 4.2ab 15 29.1 ± 4.0ab 29.5 ± 4.0abc 30 24.6 ± 4.6ab 23.6 ± 4.5ab S25 0 14.7 ± 4.3c 14.0 ± 4.2c 15 16.7 ± 4.0c 15.8 ± 4.0d 30 15.1 ± 4.6c 14.5 ± 4.5b S25DMF 0 23.0 ± 4.3abc 22.3 ± 4.2abc 15 27.2 ± 4.0abc 25.9 ± 4.0abcd 30 22.5 ± 4.6ab 21.7 ± 4.5ab S50 0 19.3 ± 4.3bc 18.4 ± 4.2bc 15 21.3 ± 4.0bc 20.5 ± 4.0bcd 30 23.2 ± 4.6ab 22.2 ± 4.5ab S50DMF 0 33.3 ± 4.3a 32.1 ± 4.2a 15 36.1 ± 4.0a 34.8 ± 4.0a 30 33.8 ± 4.6a 32.8 ± 4.5a S100 0 16.8 ± 4.3c 16.0 ± 4.2c 15 19.8 ± 4.0c 18.8 ± 4.0cd 30 19.2 ± 4.6b 18.4 ± 4.5b S100DMF 0 33.5 ± 4.3a 32.0 ± 4.2a 15 34.4 ± 4.0a 32.1 ± 4.2ab 30 35.2 ± 4.6a 33.9 ± 4.5a Rows and columns without common superscripts differed (p < 0.05) Clinical Theriogenology 2022; 14: 359 Table 3. Postthaw (mean ± SEM) VCL, VSL, VAP um/s, ALH, BCG and HAC at 0, 15, and 30 minutes of stallion sperm frozen with several sucrose concentrations +/- DMF (2%) and BSA (1%) Treatment Time (minutes) VCL (µm/s) VSL (µm/s) VAP (µm/s) ALH um BCF (Hz) HAC (rad) DMF 0 27.3 ± 3.2a 20.0 ± 2.3a 20.7 ± 2.4a 0.7 ± 0.1a 1.6 ± 0.2 0.2 ± 0.1a 15 23.3 ± 3.0bc 17.4 ± 2.5bc 18.0 ± 2.6abc 0.6 ± 0.1ab 1.6 ± 0.2ab 0.2 ± 0.0ab 30 22 ± 3.3ab 17.0 ± 2.7abc 17.5 ± 2.7ab 0.5 ± 0.1ab 1.6 ± 0.2 0.1 ± 0.0abc S25 0 13.9 ± 3.2b 10.4 ± 2.3b 10.7 ± 2.4b 0.4 ± 0.1b 1.5 ± 0.2 0.1 ± 0.1b 15 13.9 ± 3.0d 10.2 ± 2.5c 10.6 ± 2.6c 0.4 ± 0.1c 1.1 ± 0.2b 0.1 ± 0.0c 30 14.2 ± 3.3c 11.1 ± 2.7c 11.3 ± 2.7c 0.4 ± 0.1b 1.4 ± 0.2 0.1 ± 0.0c S25DMF 0 20.9 ± 3.2ab 15.5 ± 2.3ab 16.0 ± 2.4ab 0.5 ± 0.1ab 1.6 ± 0.2 0.1 ± 0.1ab 15 23.6 ± 3.0abc 17.3 ± 2.5bc 18.0 ± 2.6abc 0.6 ± 0.1ab 1.5 ± 0.2ab 0.2 ± 0.0abc 30 18.3 ± 3.3ab 11.1 ± 2.7abc 14.5 ± 2.7bc 0.5 ± 0.1ab 1.6 ± 0.2 0.1 ± 0.0bc S50 0 14.0 ± 3.2b 10.1 ± 2.3b 10.5 ± 2.4b 0.4 ± 0.1b 1.3 ± 0.2 0.1 ± 0.1b 15 19.4 ± 3.0bcd 15.5 ± 2.5bc 15.8 ± 2.6bc 0.5 ± 0.1bc 1.5 ± 0.2ab 0.1 ± 0.0bc 30 27.3 ± 3.3a 20.7 ± 2.7ab 21.0 ± 2.7a 0.6 ± 0.1a 1.7 ± 0.2 0.2 ± 0.0ab S50DMF 0 27.1 ± 3.2a 19.8 ± 2.3a 20.5 ± 2.4a 0.7 ± 0.1a 1.7 ± 0.2 0.2 ± 0.1a 15 32.4 ± 3.0a 24.9 ± 2.5a 25.6 ± 2.6a 0.7 ± 0.1a 1.8 ± 0.2a 0.2 ± 0.0a 30 16.6 ± 3.3c 12.7 ± 2.7bc 12.4 ± 2.7c 0.5 ± 0.1ab 1.4 ± 0.2 0.1 ± 0.0c S100 0 13.8 ± 3.2b 9.3 ± 2.3b 9.4 ± 2.4b 0.4 ± 0.1b 1.2 ± 0.2 0.1 ± 0.1b 15 16.4 ± 3.0cd 11.7 ± 2.5c 11.2 ± 2.6c 0.5 ± 0.1bc 1.4 ± 0.2ab 0.1 ± 0.0bc 30 16 ± 3.3c 11.2 ± 2.7c 11.6 ± 2.7c 0.4 ± 0.1ab 1.3 ± 0.2 0.1 ± 0.0c S100DMF 0 27.8 ± 3.2a 18.0 ± 2.3a 19.0 ± 2.4a 0.8 ± 0.1a 1.6 ± 0.2 0.2 ± 0.1a 15 27.6 ± 3.0ab 20.2 ± 2.5ab 20.8 ± 2.5ab 0.7 ± 0.1a 1.7 ± 0.2a 0.2 ± 0.0a 30 29 ± 3.3a 21.4 ± 2.7a 22.1 ± 2.7a 0.6 ± 0.1a 1.6 ± 0.2 0.2 ± 0.0a Rows and columns without common superscripts differed (p < 0.05) 0 5 10 15 20 25 30 35 40 45 50 Control S100 S100DMF S25 S25DMF S50 S50DMF M ot ili ty (% ) Treatment Progressive motility 0 min 15 min 30 min a a ab abc bc c c a ab abc abcd bcd cd d a a ab abab b b Figure 1. Postthaw (mean ± SEM) TM at 0, 15, and 30 minutes of stallion sperm frozen with several sucrose concentrations, +/- DMF and BSA; bars without common letters within treat- ment groups differed (p < 0.05). 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 DMF S100 S100DMF S25 S25DMF S50 S50DMF In ta ct Treatment Plasma membrane a ababc bccc c Figure 2. Postthaw (mean ± SEM) PM at 0, 15, and 30 min- utes of stallion sperm frozen with several sucrose concentra- tions, +/- DMF and BSA; bars without common letters differed (p < 0.05) among treatment groups. There was an effect (p < 0.05) of treatment on postthaw sperm PMI and ACR integrity. Postthaw PMI integrity of sperm fro- zen in treatment groups S100DMF, S50DMF, and S100 were higher than Control, S25, S25DMF, and S50 (Figure 3). As sucrose concentrations increased, postthaw intact PMI integ- rity increased, regardless of DMF presence (Figure 3). Post- thaw sperm ACR integrity in treatment groups S100, S50, S25, S100DMF, and S50DMF was higher than sperm frozen in Con- trol (Figure 4). Clinical Theriogenology 2022; 14: 360 0 5 10 15 20 25 30 35 40 45 50 Control S100 S100DMF S25 S25DMF S50 S50DMF M ot ili ty (% ) Treatments Total motility 0 min 15 min 30 min ab a a bc c c a a ab abc bc bc c a a bc bcbc c c abc Figure 3. Mean (± SEM) postthaw intact plasma membrane (PMI) of stallion sperm frozen with different sucrose concen- trations +/- DMF (2%) and BSA (1%); bars without common letters within treatments groups differed (p < 0.05). 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 DMF S100 S100DMF S25 S25DMF S50 S50DMF In ta ct Treatment Acrosome a a a ab ab bc c Figure 4. Mean (± SEM) postthaw intact acrosome (ACR) of equine sperm frozen with different sucrose concentrations +/- DMF and BSA; bars without common letters differed (p < 0.05) among treatment groups Discussion Freezing extender containing sucrose (50 and 100 mM), BSA, and DMF improved postthaw sperm integrity compared to sperm frozen in a freezing extender containing DMF only (Control). Postthaw sperm total and progressive motility, and kinematics of semen frozen in extenders containing sucrose and BSA in association with DMF (S50DMF and S100DMF) were either higher or similar to Control. Osmolality of the extenders in treatment groups S50DMF (657 mOsm kg-1), S100DMF (769 mOsm kg-1) and DMF (635 mOsm kg-1) were higher than the 500 mOsm kg-1 reported osmotic tolerance threshold for stallion sperm to maintain motility.20 Neverthe- less, the reported20 osmotic tolerance threshold GLY and use of DMF in our study may have reduced the osmotic stress suf- fered by the sperm due to its lower molecular weight (MW = 73.10 g/mol).6 There was no interaction between sucrose and permeable cryoprotectants (dimethylacetamide, DMF or GLY) preserving PMI sperm integrity during freezing stallion semen,16 possibly by the presence of egg yolk (10%) in the freezing extender. We did not use egg yolk and higher concentrations of sucrose (S100DMF and S50DMF) better preserved plasma membrane integrity. Furthermore, that S50DMF treatment group PMI in- tegrity was higher than the S50 treatment and not different than S100DMF, suggested that sucrose positively interacted with DMF. Beneficial effects of this association may be due to the following. Sucrose associated with membrane phos- pholipids head, stabilized membrane bilayer during freez- ing21 via electrostatic binding of saccharide hydroxyl groups to phosphate groups on the membrane lipid head,22 reduced ice crystal formation23-25 and provided an energy substrate.26 Additionally, DMF stabilized the membrane bilayer,21 modu- lated cell dehydration,24 and decreased osmotic tension of the unfrozen fraction.27 Furthermore, BSA antioxidant properties prevented lipid peroxidation that is involved in motility loss following cryopreservation.28 During fertilization, viable sperm undergo a morphologi- cal change known as capacitation.1 Freezing-thawing stal- lion sperm induce capacitation-like changes, increasing the proportion of sperm with reacted acrosomes.29 In our study, postthaw acrosome integrity was better preserved with su- crose. Similar to results of PMI integrity, as sucrose concen- trations increased, postthaw acrosome integrity preservation improved. However, our results did not appear to suggest that sucrose and DMF interacted to improve acrosome protection during freezing-thawing of stallion sperm. Minimum acceptable postthaw motility of frozen semen for commercial use is 30%.2,30 Extenders S50DMF and S100DMF achieved a postthaw sperm total and progressive motility > 30% immediately and it was maintained until 30 minutes af- ter thawing. Additionally, S50DMF, and S100DMF extenders had higher postthaw intact PMI and ACR. This suggested that freezing extenders containing sucrose and BSA in association with DMF may be an alternative for freezing stallion semen. Our study had some limitations. Freezing extender containing sucrose (50 and 100 mM), BSA, and DMF appeared accept- able protecting stallion sperm during freezing-thawing; how- ever, the optimal concentration of sucrose for stallion sperm cryopreservation must be determined. Although extenders with sucrose, BSA, and DMF, achieved a postthaw motility above the recommended 30%, the fertilization capacity of the thawed sperm is unknown. Further research must investigate the optimal concentration of sucrose and fertilizing capacity after cryopreservation of stallion sperm with sucrose extend- ers. Combining sucrose BSA and DMF in the freezing extender may be an alternative for cryopreservation of stallion semen. As concentrations of sucrose increased in combination with DMF, the postthaw PMI and ACR integrity improved. Acknowledgement Authors thank Dr. Karen Kind and staff at University of Ade- laide Equine Health and Performance Centre for their assis- tance. 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