Equilibration-free cryopreservation of beef and bison semen

1 1 Equilibration-free cryopreservation of beef and bison semen 2 3 Short title: Semen cryopreservation without equilibration 4 5 Yang S1,2, Rajapaksha K2, Zwiefelhofer E1,#a, Adams GP1 and Anzar M2* 6 7 1 Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, 8 University of Saskatchewan, Saskatoon, Canada 9 10 2 Agriculture and Agri-Food Canada, Saskatoon Research and Development Center, Saskatoon, 11 Canada 12 13 #aCurrent Address: STgenetics, 22575 Highway 6 S, Navasota, Texas, United States of America 14 15 * Corresponding author 16 E-mail: [email protected] & [email protected] 17 18 These authors contributed equally to this work. 19 20 CONFLICT OF INTEREST: The authors have declared that no competing interests exist. 21 22 23 24 25 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 2 26 Abstract 27 Conventional semen cryopreservation involves equilibration at 4°C and optimum freezing rates. 28 We hypothesized that a cholesterol-based semen extender obviates the need for equilibration, 29 minimizing total processing time for semen cryopreservation. Experiments were conducted to 30 determine the effects of semen extender (egg yolk- or cholesterol-based) and freezing method 31 (routine or fast) on post-thaw sperm characteristics and fertility of beef and bison semen. In 32 Experiment 1, beef semen diluted in tris-egg yolk-glycerol (TEYG) or cholesterol-cyclodextrin 33 tris-glycerol (CCTG) extender underwent routine or fast freezing method. Cholesterol from 34 animal and plant origins were compared. The routine method included 90-min equilibration at 35 4°C and routine freezing (RE-RF, total time 97 min) whereas the fast method included no 36 equilibration and fast freezing (NE-FF, total time 14 min). Post-thaw sperm quality was assessed 37 by CASA, and in vitro fertilization. Post-thaw sperm motility was not affected by the origin of 38 cholesterol (animal or plant), but was lowest in the TEYG NE-FF group (24% vs 43-51%, P < 39 0.05). In vitro cleavage and blastocyst development rates did not differ between RE-RF and NE- 40 FF groups. In Experiment 2, bison semen was diluted in TEYG or plant-CCTG extender and 41 frozen as in Experiment 1. Post-thaw sperm motility was lowest in the TEYG NE-FF group 42 (10% vs 39-51%, P < 0.05). In Experiment 3, beef semen diluted in TEYG or plant-CCTG 43 extender underwent either a routine (RE-RF) or modified freezing (NE-RF, total time 25 min) 44 method. Post-thaw sperm characteristics did not differ between extenders but were greater using 45 routine freezing (RE-RF) compared to the modified method of freezing (NE-RF). Pregnancy 46 rates were similar between extenders (TEYG vs plant-CCTG) using the modified freezing 47 method without equilibration and insemination at 72 h after progesterone device removal. In .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 3 48 conclusion, beef and bison semen diluted in cholesterol-based extender may be cryopreserved 49 without equilibration. (Words 297/300) 50 KEY WORDS: semen; equilibration; freezing rate; beef; bison 51 52 Introduction 53 Mammalian semen cryopreservation procedures include dilution in cryoprotective extenders, 54 cooling to 4°C, and freezing below 0°C. Semen extenders increase both ejaculate volume and 55 sperm longevity. During initial cooling to 4°C, sperm plasma membranes biochemically interact 56 with extender constituents, specifically referred to as ‘equilibration’ [1]. Mammalian sperm 57 plasma membranes undergo lipid-phase transitions between 18° to 14°C, known as ‘cold shock’ 58 [2-4], characterized by lateral movement of phospholipids which increase membrane 59 permeability to ions, and damages sperm membranes irreversibly [5,6]. Upon ejaculation, binder 60 of sperm proteins in seminal plasma causes efflux of phospholipids and cholesterol from plasma 61 membranes. Egg yolk in extender mitigates the cold shock effect by a mechanism that involves 62 sequestering binder of sperm proteins in seminal plasma [7,8] and binding with sperm plasma 63 membranes [9], thus stabilizing sperm against membrane lipid-phase transitions and replacing 64 phospholipids lost during freezing and thawing [10-13]. 65 After dilution in extender, semen is commonly held at 4°C for 90-120 min to achieve 66 equilibrium between components in semen extender and sperm plasma membranes. With egg 67 yolk-glycerol extenders, the equilibration time is commonly ≥90 min before freezing below 0°C 68 [14]. However, a wide range of equilibration time has been reported in literature; long 69 equilibration time (>12 h) provided better post-thaw sperm quality than short time [1,15-19]. In a .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 4 70 typical semen cryopreservation protocol, the prerequisite equilibration step prolongs the total 71 processing time and negatively impacts the efficiency of commercial semen production centers. 72 Earlier work demonstrated that exogenous cholesterol is incorporated into sperm plasma 73 membranes within 15 min of incubation in the presence of egg yolk components, and improved 74 post-thaw sperm motility in beef and bison bulls [20-22]. However, the use of egg yolk, an 75 animal product, in semen extender raises biosecurity concerns regarding transmission of 76 infectious diseases. Moreover, egg yolk has undefined composition and varies from batch to 77 batch. Cholesterol, commonly from sheep’s wool, is known to modulate membrane fluidity, 78 increase membrane stability, and minimize lipid-phase transitions during cooling [23]. 79 Alternatively, cholesterol may be sourced from plants to minimize biosecurity risks associated 80 with animal products and have a defined composition. Cyclodextrins act as carriers to deliver 81 cholesterol to sperm plasma membrane [22-24]. In this regard, we developed a novel cholesterol- 82 based extender as an alternative to conventional egg yolk extender for cryopreservation of beef 83 and bison semen to mitigate biosecurity risks [11,25]. 84 After equilibration, sperm survival below 0°C depends primarily on minimizing two 85 physico-chemical effects: intracellular ice crystal formation and high solute concentrations [26]. 86 If cell freezing occurs at an overly fast rate, incomplete dehydration leads to intracellular ice 87 formation of residual water, causing physical damage to the cell membrane and organelles. 88 Conversely, if the freezing rate is too slow, prolonged intracellular dehydration leads to high 89 solute concentrations (solution effect) and toxicity. Glycerol, a low-molecular weight penetrating 90 cryoprotectant, readily crosses the plasma membrane, binds with free water and helps in 91 preventing ice crystal formation, and can be added either before or after cooling [1,25,27,28]. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 5 92 Therefore, total processing time may be further reduced by implementing a fast freezing rate 93 below 0°C. 94 North American wood and plains bison are threatened by genetic isolation and disease, 95 and concerted efforts are underway to rescue the species by establishing a bison biobank with the 96 use of germplasm in assisted reproductive technologies [25,29-31]. However, semen collection 97 from free-roaming bison under wild conditions poses logistical concerns related to 98 cryopreservation processing time and facilities. There is a need for a simple and quick method of 99 semen cryopreservation to enable collection and processing under remote field conditions and 100 during extremely cold months. 101 In the present study, attempts were made to develop a short cryopreservation protocol for 102 bovine and bison semen by removing the most time-consuming equilibration step, and by 103 freezing at a faster rate. We hypothesized that a cholesterol-based semen extender obviates the 104 need for equilibration minimizing total processing time for semen cryopreservation. The specific 105 objectives of this study were to determine the effects of semen extender (egg yolk- or 106 cholesterol-based), origin of cholesterol (animal or plant), and freezing method (routine or fast) 107 on post-thaw sperm quality and fertility of beef and bison semen. 108 Materials and Methods 109 Beef cattle and bison bulls were housed separately (5 km apart) under similar management and 110 nutrition conditions at the Livestock and Forage Center of Excellence, University of 111 Saskatchewan, Saskatoon. Animal procedures were conducted in accordance with the Canadian 112 Council on Animal Care and approved by the University of Saskatchewan Animal Care 113 Committee (Animal Use Protocol #20100150). In all experiments, semen was collected by .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 6 114 electroejaculation (Pulsator IV Auto Adjust; Lane Manufacturing Inc., Denver, CO, USA). After 115 collection, semen was transported to the Cryobiology Laboratory, Westgen Research Suite, 116 University of Saskatchewan, within 2 h at 32°C. Ejaculates were evaluated by computer-assisted 117 sperm analyzer (CASA; Sperm Vision 3.0, Minitube Canada, Ingersoll, ON, Canada), as 118 reported earlier [20,32]. Ejaculates possessing sperm concentration >200×106 sperm/mL and 119 total motility >60% were pooled to minimize bull-to-bull differences, for further processing. All 120 chemicals were purchased from Sigma-Aldrich (Oakville, ON, Canada) unless otherwise stated. 121 Semen extender preparation 122 Cholesterol-cyclodextrin complex (CC) was prepared as previously described [21]. Solution A 123 was prepared by dissolving 200 mg cholesterol of animal-origin (sheep wool; Cat# C8667) or 124 plant-origin (PhytoChol®, Wilshire Technologies, Princeton, NJ, USA; Cat# 57-88-5) in 1 mL 125 chloroform. Solution B was prepared by dissolving 1 g methyl β-cyclodextrin (Cat# C4555) in 2 126 mL methanol. Solution A (0.45 mL) was then added to Solution B (2 mL) and mixed until the 127 solution became homogenous. The mixture was then poured into a glass petri dish and dried 128 under a gentle stream of nitrogen gas. The resulting crystals were desiccated overnight and 129 stored in a glass bottle in desiccator at 22°C until used. On the day of experiment, a working 130 solution of CC (50 mg/mL) was prepared in tris-citric acid (TCA) buffer containing tris-base 131 3.03% wt/vol, citric acid monohydrate 1.74% wt/vol, and fructose 1.2% wt/vol (pH 7.1) in Milli- 132 Q distilled water and used immediately. 133 Tris-glycerol (TG; 2×) extender was prepared by adding glycerol (14% v/v), gentamycin 134 sulfate (1 mg/mL), tylosin (200 µg/mL; Tylan Soluble, Elanco, Guelph, ON, Canada), and 135 lincomycin-spectinomycin (600/1200 µg/mL; Linco-Spectin, Pfizer Animal Health, Kirkland, 136 QC, Canada) to TCA buffer, and stored at -20°C. TG (2×) extender was added to CC treated .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 7 137 semen (1:1) to achieve the final concentrations of glycerol (7% v/v), gentamycin sulfate (500 138 µg/mL), tylosin (100 µg/mL), and lincomycin-spectinomycin (300/600 µg/mL). The combined 139 extender will be referred to as ‘CCTG’ hereafter. 140 A conventional tris-egg yolk-glycerol (TEYG) extender was prepared by adding glycerol 141 (7%, v/v), egg yolk (20%, v/v), gentamycin sulfate (500 µg/mL), tylosin (100 µg/mL), and 142 lincomycin-spectinomycin (300/600 µg/mL) in TCA buffer. The final extender was centrifuged 143 at 12,000× g for 15 min at 4°C. The supernatant was recovered and stored at -20°C. All frozen 144 media were thawed to 37°C on the day of the experiment. 145 Experiment 1: Effect of extender, origin of cholesterol and freezing 146 method on post-thaw sperm quality and in vitro fertility of beef bulls 147 Experiment 1a (Simmental bulls): 148 Semen was collected from 5 Simmental bulls on 5 different dates (replicates) to determine the 149 effects of semen extender (egg yolk- or cholesterol-based), source of cholesterol (animal or 150 plant) and freezing method (routine or fast) on post-thaw sperm motility, and plasma membrane 151 and acrosome integrity. Pooled ejaculates were divided into three extender groups: TEYG, 152 animal-CCTG, or plant-CCTG, as previously described [11,25]. For the TEYG group, semen 153 was diluted to 50×106 sperm/mL with extender (32°C) and held at 22°C for 15 min. For animal- 154 and plant-CCTG groups, semen was initially diluted to 100×106 sperm/mL with TCA buffer at 155 32°C, treated with 1 mg animal- or plant-CCTG/ml semen at 22°C for 15 min, and then diluted 156 1:1 with TG (2×) to achieve a final sperm concentration of 50×106/mL [25]. The final 157 concentration of animal- or plant-CC was 0.5 mg/ml semen. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 8 158 Each TEYG- and CCTG-diluted batch of semen underwent either a routine (routine 159 equilibration-routine freezing; RE-RF) or fast freezing (no equilibration-fast freezing; NE-FE) 160 method (Table 1). In the routine method, 15-ml tubes of extended semen were placed in a 500 161 mL glass beaker containing water (22°C) and cooled at 4°C for 90 min (equilibration), then filled 162 into 0.5 mL straws and frozen using a routine freezing curve (i.e., -3°C/min from 4°C to -10°C, - 163 40°C/min from -10°C to -80°C) in a programmable cell freezer (ICE-CUBE 14-S; Sy-lab 164 Version 1.30, Gerate GmbH, Neupurkdersdof, Austria) [11]. In the fast method, diluted semen 165 was immediately filled into 0.5 mL straws at 22°C, and frozen directly in a programmable cell 166 freezer without equilibration at 4°C (i.e., -1°C/min from 22°C to 10°C, and -40°C/min from 167 10°C to -80°C). After reaching -80°C in both freezing methods, semen straws were plunged into 168 liquid nitrogen, and stored until thawing. 169 For post-thaw sperm analysis, two straws from each treatment group were thawed at 170 37°C for 1 min and contents were pooled to minimize straw-to-straw variation. Sperm motility 171 was determined at 0 and 2 h post-thaw by CASA using bull settings [16,32], and in vitro 172 fertilization potential of sperm in all treatment groups was determined [25,33,34]. Both CASA 173 and IVF procedures are described under the section below (Semen assays). 174 Experiment 1b (Angus bulls): 175 Semen was collected from 5 Angus bulls on 7 different dates (replicates) and pooled, as 176 described above. Since the effect of CC origin (animal- or plant-derived) was not significant for 177 any endpoint in Experiment 1a, subsequent experiments were conducted using plant-CCTG 178 extender for enhanced biosecurity. The effects of semen extender (egg yolk or plant-CCTG) and 179 freezing method (routine or fast) on post-thaw sperm motility and structural characteristics .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 9 180 (intact plasma and acrosome membranes) were evaluated at 0 and 2 h, by CASA and flow 181 cytometer respectively, as described below (Semen assays). 182 Table 1. Duration of routine and fast freezing methods for cryopreservation of beef and bison 183 semen (Experiments 1 & 2). Stage of cryopreservation Routine method (RE-RF)* Fast method (NE-FF)* Cooling/equilibration (time) 22ºC to 4ºC (90 min) 22°C to 10°C @ -1°C/min (12 min) Freezing ramp 1 (time) 4ºC to -10ºC @ -3°C/min (~5 min) 10°C to -80°C @ -40°C/min (~2 min) Freezing ramp 2 (time) -10ºC to -80ºC @ -40°C/min (~2 min) - Total processing time ~97 min ~14 min 184 *RE-RF: routine equilibration-routine freezing; NE-FF: no equilibration-fast freezing. 185 186 Experiment 2: Effect of extender and freezing method on post-thaw 187 sperm quality of bison bulls 188 Semen was collected from 5 bison bulls on 5 different dates and pooled, as described above. 189 Pooled semen was diluted in either TEYG or plant-CCTG extender and underwent routine or fast 190 method (Table 1). As in Experiment 1, post-thaw sperm motility, and plasma membrane and 191 normal acrosome were assessed by CASA and flow cytometry respectively, at 0 and 2 h, as 192 described below (Semen assays). 193 194 Experiment 3: Effect of extender and modified freezing method on 195 post-thaw sperm quality and in vivo fertility of beef bulls .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 10 196 Semen was collected from 6 Angus bulls on 10 different dates (replicates) and pooled, as 197 described above. Pooled semen was diluted in either TEYG or plant-CCTG extender and frozen 198 using either a routine (RE-RF) or modified (no equilibration-routine freezing, NE-RF) method 199 (Table 2). Semen straws were deep-frozen using a routine freezing curve, as described in 200 Experiment 1. In the modified group, the extended semen was loaded immediately into 0.5 mL 201 straws at 22°C and cooled from 22°C to 4°C @ -1°C/min using a programmable cell freezer 202 (without equilibration). Below 0°C, the routine freezing curve was used, as described above. 203 Post-thaw sperm motility, and plasma membrane integrity and normal acrosomes were 204 determined using CASA and flow cytometry respectively, at 0 and 2 h, as described below 205 (Semen assays). This experiment was repeated on ten pooled ejaculates (replicates) from four 206 Angus bulls, on different dates. 207 Table 2. Duration of routine and modified freezing methods of for cryopreservation of beef 208 semen (Experiment 3). Stage of cryopreservation Routine method (RE-RF)* Modified method (NE-RF)* Cooling (time) 22ºC to 4ºC (90 min) 22ºC to 4°C @ -1°C/min (18 min) Freezing ramp 1 (time) 4ºC to -10ºC @ -3°C/min (~5 min) 4ºC to -10ºC @ -3°C/min (~5 min) Freezing ramp 2 (time) -10ºC to -80ºC @ -40°C/min (~2 min) -10ºC to -80ºC @ -40°C/min (~2 min) Total processing time ~97 min ~25 min 209 *RE-RF: routine equilibration-routine freezing; NE-RF: no equilibration-routine freezing. 210 211 212 213 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 11 214 Fertility trial 1 215 A fertility trial was conducted to compare the effect of routine semen processing (TEYG 216 extender and routine freezing [RE-RF]) vs modified semen processing (plant-CCTG extender, 217 modified freezing [NE-RF]) on pregnancy rate after fixed-time insemination. In addition, two 218 different intravaginal progesterone devices (CIDR [Zoetis Canada Inc., Kirkland, QC, Canada] 219 or PRID-Delta [Ceva Animal Health, Cambridge, ON, Canada]) were compared as part of a 220 separate study. Lactating Angus-cross cows (n = 206) at random stages of the estrous cycle and 221 at least 40 days post-partum were divided into 3 replicates and synchronized using a standard 5- 222 day progesterone-based protocol with either a CIDR (n = 105) or PRID-Delta (n = 101; Fig. 1). 223 On Day 0, cows were given GnRH (100 µg gonadorelin im, Fertilin) and an intravaginal 224 progesterone device. The progesterone device was removed on Day 5, and cows were given a 225 luteolytic dose of PGF2α (500 µg cloprostenol im, Bioestrovet; Vetoquinol, Lavaltrie, QC, 226 Canada) at the time of device removal and again 24 h later. On Day 8 (72 h after PGF2α 227 treatment), cows were treated with GnRH (100 µg gonadorelin im, Fertilin) and inseminated with 228 a single dose of TEYG routine (n = 104) or plant-CCTG modified (n = 102) semen, randomly 229 distributed between synchronization treatments. Ovulation was confirmed by transrectal 230 ultrasonography at the time of insemination or subsequent examinations 24 and 48 h post- 231 insemination (MyLab Five, Esaote North America Inc, Fishers, IN, USA), defined as the 232 disappearance of a large follicle between successive examinations or detection of a new CL. 233 Pregnancy was diagnosed by transrectal ultrasonography at 27 to 33 days after insemination. The 234 pregnancy rate was calculated as number pregnant out of those inseminated. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 12 235 236 Fig 1. Fixed-time artificial insemination synchronization protocol. Cows were treated with 237 GnRH at the time of placement of a progesterone-releasing intravaginal device (CIDR, n = 105 238 or PRID-Delta, n = 101). A luteolytic dose of PGF2α was given at the time of device removal 239 and again 24 h later. Cows were treated with GnRH at the time of insemination 72 h after device 240 removal using semen processed in 2 different ways (TEYG routine [RE-RF], n = 104 or plant- 241 CCTG modified [NE-RF], n = 102; Fertility trial 1). 242 Abbreviations: AI, Artificial insemination; CIDR, Controlled internal drug release; GnRH, 243 Gonadotropin-releasing hormone; PGF, Prostaglandin F2α; PRID, Progesterone-releasing 244 intravaginal device; US, Ultrasound. 245 246 Fertility trial 2 247 A follow-up 2×2 fertility trial was conducted to determine the effect of modified semen (no 248 equilibration-routine freezing, NE-RF) extended in either TEYG or plant-CCTG extenders, and 249 insemination time (60 h or 72 h after prostaglandin treatment) on pregnancy rate after fixed-time 250 artificial insemination. Lactating multiparous Hereford-cross cows (n = 106) at random stages of 251 the estrous cycle and at least 45 days post-partum, were synchronized (Fig. 2). A progesterone- 252 releasing intravaginal device (CIDR, Vetoquinol, Lavaltrie, QC, Canada) was inserted on Day 0 253 and removed on Day 5. A luteolytic dose of PGF2α (500 µg cloprostenol im, Estroplan, 254 Vetoquinol, Lavaltrie, QC, Canada) was given at the time of device removal. Cows were then 255 assigned randomly to be inseminated at either 60 h (n = 65) or 72 h (n = 41) after PGF2α 256 treatment with either TEYG modified (n = 52) or plant-CCTG modified (n = 54) semen group, 257 and treated with GnRH (100 µg gonadorelin im, Fertilin, Vetoquinol, Lavaltrie, QC, Canada) at 258 the time of insemination. Ovulation status and pregnancy rates were determined as described in 259 Fertility trial 1. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 13 260 Fig 2. Fixed-time artificial insemination synchronization protocol. A progesterone-releasing 261 intravaginal device (PRID) was placed for a period of 5 days and a luteolytic dose of PGF2α was 262 given at the time of device removal. Cows were treated with GnRH at the time of insemination at 263 either 60 h (n = 65) or 72 h (n = 41) after device removal, using modified (NE-RF) semen diluted 264 TEYG (n = 52) or plant-CCTG (n = 54) extenders (Fertility trial 2). 265 Abbreviations: AI, Artificial insemination; GnRH, Gonadotropin-releasing hormone; PGF, 266 Prostaglandin F2α; PRID, Progesterone-releasing intravaginal device; US, Ultrasound. 267 268 Semen assays 269 Computer-assisted sperm analysis 270 In all experiments, a semen sample (2.5 µl) was placed on a prewarmed (37°C) Leja 271 chamber slide (depth 20 µm; Leja Products B.V. Nieuw-Vennep, Netherlands) and analyzed 272 using a computer-assisted sperm analyzer (CASA; Sperm Vision1 3.0, Minitube Canada). A 273 minimum 200 sperm in 7 fields were assessed for total motility (%; all moving sperm) and 274 progressive motility (%; sperm moving in straight-line, i.e., more than 10 µm radius with a speed 275 of > 4.5 µm/s). 276 Flow cytometer analysis 277 In Experiments 1b, 2 and 3, plasma and acrosome membrane integrity were assessed by 278 flow cytometer (CyFlow Space, Partec GmbH, Münster, Germany) using propidium iodide (PI; 279 stock 2.4 mM in water) and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA; stock 1 280 mg/mL in PBS) fluorescent markers, as previously described [16]. Briefly, 1×106 sperm from 281 each treatment group were diluted in 500 µL TCA buffer and incubated with 5 µL PI and 1 µL 282 FITC-PNA at 22°C in the dark for 20 min. Sperm were fixed by adding 10 µL 10% .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 14 283 formaldehyde (v/v). Data acquired by FloMax software (version 2.4, Partec GmbH, Münster, 284 Germany) revealed four different sperm populations based on plasma and acrosome membrane 285 integrities: i) sperm with intact plasma membrane and intact acrosome (PI-/FITC-PNA-, intact), 286 ii) sperm with intact plasma membrane and reacted acrosomes (PI-/FITC-PNA+, partially intact), 287 iii) sperm with compromised plasma membrane and intact acrosomes (PI+/FITC-PNA-, partially 288 intact), and iv) sperm with compromised plasma membrane and reacted acrosome (PI+/FITC- 289 PNA+; compromised). Sperm population with an intact plasma membrane and intact acrosome 290 (PI-/FITC-PNA-; intact) was selected for further statistical analysis. 291 In all experiments, a stress test was conducted by holding frozen-thawed semen at 37°C 292 for 2 h and analyzed for sperm motility and/or structural characteristics with CASA and flow 293 cytometer respectively. 294 In vitro fertilization 295 In Experiment 1a, in vitro fertilization potential of frozen-thawed semen extended in 296 TEYG, animal-CCTG, or plant-CCTG extender, and frozen with either routine or fast method 297 were determined. Cattle ovaries were collected from a slaughterhouse near Calgary, Alberta and 298 transported by air to the Cryobiology Laboratory within 8 h, at 22˚C. In vitro maturation, 299 fertilization and embryo culture were performed, as previously described [25,34,35]. Briefly, 300 cumulus-oocyte complexes (COC) were aspirated from follicles (3-8 mm) and identified under a 301 stereomicroscope (10×). COC with uniform cytoplasm containing >3 layers of cumulus cells 302 were selected for further processing. COC were washed (3×) in maturation medium (5% calf 303 serum v/v, 0.5 µg/mL FSH, 5 µg/mL LH and 0.05 µg/mL gentamycin in TCM199). 304 Approximately 20 COC were pipetted into a 100 µL droplet of maturation medium and .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 15 305 incubated under mineral oil at 38.5°C and 5% CO2 in air, for 22 h. Frozen-thawed semen 306 samples were centrifuged (2000× g) through Percoll density gradients (45% and 90%) in a 15 307 mL conical tube for 15 min and pellet was diluted to 3×106 sperm/mL with Brackett-Oliphant 308 (BO) fertilization medium [25]. Mature COC were washed (3×) with BO medium containing 309 10% (w/v) bovine serum albumin (BSA), placed in 100 µL droplets possessing 300,000 sperm, 310 and incubated at 38.5°C and 5% CO2 in air, for 18 h (Day 0 = day of IVF). After incubation, the 311 presumptive zygotes were denuded by repeated pipetting while washing (3×) in 100 µL droplets 312 of Charles Rosenkrans1 + amino acids culture media (CR1aa) + 5% v/v calf serum under mineral 313 oil at 38.5°C, 5% CO2, 5% O2 and 90% N2. Cleavage and blastocyst rates were evaluated on Day 314 2 and 8, respectively, and reported based on the number of oocytes submitted to IVF. This trial 315 was conducted four times on different dates (replicates). 316 Statistical analysis 317 Values are expressed as mean±standard error of the mean (SEM) unless otherwise stated. 318 In Experiment 1, 3 × 2 factorial analysis was used to study the effect of extenders (TEYG, 319 animal-CCTG and plant-CCTG), freezing methods (routine and fast) and their interactions on the 320 post-thaw sperm characteristics. In Experiments 2 and 3, factorial analysis (2 × 2) was used to 321 determine the effect of extenders and freezing methods. If main effects or their interaction were 322 significant (P ≤ 0.05), means were compared by Tukey’s test. In vitro cleavage and blastocyst 323 rates, and in vivo pregnancy rates were compared among groups by binomial generalized linear 324 mixed model analysis of variance. Data analyses were performed using the R Project (R version 325 3.3.1, R Foundation for Statistical Computing, Vienna, Austria). 326 Results .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 16 327 Experiment 1: Effect of extender, origin of cholesterol and freezing 328 method on post-thaw sperm quality and in vitro fertility of beef bulls 329 Experiment 1a (Simmental bulls) 330 Extender (TEYG, animal-CCTG or plant-CCTG) × freezing method (routine or fast) 331 interaction was significant on post-thaw total and progressive motility at 0 and 2 h (Table 3). The 332 TEYG NE-FF group had the lowest post-thaw total and progressive motility compared to other 333 treatment groups. Post thaw motility did not differ between animal- and plant-origin cholesterol. 334 335 336 Table 3. Post-thaw sperm motility of beef semen diluted in TEYG, animal-CCTG or plant- 337 CCTG extender and frozen with routine or fast freezing method. Each value represents the 338 mean±SEM of five pooled ejaculates (replicates), from five Simmental bulls (Experiment 1a). Routine method (RE-RF) Fast method (NE-FF)Sperm characteristics Time TEYG Animal- CCTG Plant- CCTG TEYG Animal -CCTG Plant- CCTG 0 48±5.0a 50±4.7a 52±5.3a 24±3.2b 47±3.3a 43±3.1aTotal motility (%) 2 51±2.6a 50±4.8a 46±7.6a 21±2.5b 45±4.7a 40±8.8a 0 38±3.5a 43±4.9a 44±5.0a 20±3.1b 38±2.3a 36±2.0aProgressive motility (%) 2 40±3.8a 43±4.5a 40±6.8a 14±2.1b 37±4.2a 32±7.3a 339 abWithin rows, values with different superscripts are different (P < 0.05). 340 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris- 341 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-FF, no equilibration-fast 342 freezing. 343 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 17 344 Following in vitro fertilization of cattle oocytes with frozen-thawed sperm, cleavage and 345 blastocyst rates ranged from 55% to 65% and 23% to 33%, respectively (Table 4). No effect of 346 extender or freezing method was detected on cleavage or blastocyst rate. 347 348 Table 4. Cleavage and blastocyst rates following in vitro fertilization of bovine oocytes with 349 beef semen diluted in TEYG, animal-CCTG or plant-CCTG extender and frozen with routine or 350 fast freezing method. Each value represents four replicates conducted on different dates 351 (Experiment 1a). Routine method (RE-RF) Fast method (NE-FF)IVF variables TEYG Animal- CCTG Plant- CCTG TEYG Animal- CCTG Plant- CCTG Cleavage rate* 58/96 (60%) 73/114 (64%) 58/105 (55%) 52/83 (63%) 83/134 (62%) 87/133 (65%) Blastocyst rate* 29/96 (30%) 35/114 (31%) 29/105 (28%) 19/83 (23%) 38/134 (28%) 44/133 (33%) 352 *Cleavage and blastocyst rates were calculated based on the number of oocytes submitted to 353 IVF. 354 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris- 355 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-FF, no equilibration-fast 356 freezing. 357 358 Experiment 1b (Angus bulls) 359 Extender × freezing method interactions were significant for all post-thaw sperm 360 characteristics at 0 and 2 h (Table 5). Once again, all post-thaw sperm characteristics were 361 lowest in the TEYG fast (NE-FF) group than in other groups. Post-thaw sperm total and 362 progressive motility, and sperm with intact plasma and acrosome membranes were greater (P < 363 0.05) in the routine (RE-RF) than the fast (NE-FF) method. Within the fast method, post-thaw .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 18 364 sperm characteristics at 0 h were significantly greater (P < 0.05) in plant-CCTG than TEYG 365 extender. Within the routine method, differences between TEYG and plant-CCTG, were not 366 significant for any sperm characteristic. 367 368 Table 5. Post-thaw sperm characteristics of beef semen diluted in TEYG or plant-CCTG 369 extender and frozen with routine or fast method. Each value represents mean±SEM of seven 370 pooled ejaculates (replicates), from five Angus bulls (Experiment 1b). Routine method (RE-RF) Fast method (NE-FF)Sperm characteristics Time (h) TEYG Plant-CCTG TEYG Plant-CCTG 0 49±2.6a 42±1.9ab 14±2.5c 32±3.4b Total motility (%) 2 29±1.6a 24±2.1a 5±0.4b 4±0.6b 0 41±3.1a 33±2.4ab 9±2.1c 24±3.0b Progressive motility (%) 2 14±1.6a 13±2.5a 1±0.1b 1±0.3b 0 57±2.7a 64±1.6a 23±1.6c 44±3.8bIntact plasma & acrosome membrane (%) 2 40±3.3b 52±2.5a 11±0.7d 22±3.5c 371 abcdWithin rows, values with no common superscript are different (P < 0.05). 372 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris- 373 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-FF, no equilibration-fast 374 freezing. 375 376 Experiment 2: Effect of extender and freezing method on post-thaw sperm 377 quality of bison bulls 378 Bison sperm in the TEYG NE-FF group also had the lowest (P < 0.05) total and 379 progressive motility, and sperm with intact plasma and acrosome membrane integrity compared 380 to other treatment groups at 0 h post-thaw (Table 6). There was no difference in post-thaw sperm 381 characteristics between TEYG and plant-CCTG in the routine group (RE-RF), at 0 and 2 h. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 19 382 Sperm extended in TEYG or plant-CCTG extenders, and underwent the fast method (NE-FF) 383 yielded lower sperm motility and sperm with intact plasma membrane integrity and normal 384 acrosomes at 2 h, compared to routine group (RE-RF, P < 0.05). 385 Table 6. Post-thaw sperm characteristics of bison semen diluted in TEYG or plant-CCTG 386 extender and frozen with a routine or fast method. Each value represents mean±SEM of five 387 pooled ejaculates (replicates), from five bison bulls (Experiment 2). Routine method (RE-RF) Fast method (NE-FF) Sperm characteristics Time (h) TEYG Plant-CCTG TEYG Plant-CCTG 0 45±1.0a 51±4.1a 10±1.2b 39±4.8a Total motility (%) 2 32±2.8a 31±2.1a 3±1.0b 3±0.4b 0 37±1.0a 45±4.4a 5±1.0b 33±4.7a Progressive motility (%) 2 26±2.0a 24±2.4a 1±0.2b 1±0.4b 0 47±1.5a 52±6.1a 17±1.9c 30±4.6bIntact plasma membrane & acrosome (%) 2 39±1.9a 42±4.7a 15±1.7b 15±2.8b 388 abcWithin rows, values with no common superscript are different (P < 0.05). 389 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris- 390 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-FF, no equilibration-fast 391 freezing. 392 393 Experiment 3: Effect of extender and modified freezing method on post-thaw 394 sperm quality and in vivo fertility of beef bulls 395 There was no effect of extender or interaction between main effects on post-thaw sperm 396 characteristics. However, sperm motility, and membrane and acrosome integrity were lower in 397 the modified than routine freezing method (P < 0.05; Table 7). .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 20 399 Table 7. Post-thaw sperm motility, and plasma membrane integrity and normal acrosomes of 400 beef semen diluted in TEYG or plant-CCTG and frozen using a routine or modified method. 401 Each value represents the mean±SEM of ten pooled ejaculates (replicates) from six Angus bulls 402 (Experiment 3). Routine method (RE-RF) Modified method (NE-RF) Sperm characteristics Time (h) TEYG Plant-CCTG TEYG Plant-CCTG 0 45±3.4a 43±1.6a 37±3.4b 37±3.4b Total motility (%) 2 33±3.8a 31±2.0a 25±4.0b 22±3.2b 0 35±3.6a 34±2.2a 26±3.5b 27±3.5b Progressive motility (%) 2 23±3.9a 21±2.2a 16±3.1b 12±3.4b 0 68±2.4a 64±3.4a 56±8.6 b 56±3.9 b Intact membranes (%) 2 56±3.6a 52±4.4a 38±9.5b 37±4.8b 403 abWithin rows, values with different superscripts are different (P < 0.05). 404 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris- 405 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-RF, no equilibration- 406 routine freezing. 407 408 In Fertility trial 1, no difference was detected between CIDR and PRID-Delta groups in 409 ovulation rate (102/105 [97%] vs 101/101 [100%] or interval to ovulation from GnRH treatment 410 (30.8±1.5 h vs 32.3±1.6 h). Pregnancy rate was greater with PRID-Delta (74/101 [73%] 411 compared to CIDR (63/105 [60%], P ≤ 0.05), and TEYG routine (RE-RF; 78/104 [75%]) 412 compared to plant-CCTG modified (NE-RF; 59/102 [58%]; Table 8). 413 414 415 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 21 416 Table 8. Pregnancy rate (number pregnant/number inseminated) in beef cattle following fixed- 417 time artificial insemination using CIDR vs PRID-Delta as a source of progesterone for ovarian 418 synchronization and using either a routine (TEYG, RE-RF) or modified (plant-CCTG, NE-RF) 419 semen processing method (Fertility trial 1). Semen processing methodSynchronization protocol TEYG routine (RE-RF) Plant-CCTG modified (NE-RF) Total CIDR 37/52 (71%)a 26/53 (49%)b 63/105 (60%)x PRID-Delta 41/52 (79%)a 33/49 (67%)a 74/101 (73%)y Total 78/104 (75%)a 59/102 (58%)b 137/206 (66%) 420 Values with different superscripts within a row (a,b) and column (x,y) are different (P < 0.05). 421 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris- 422 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-RF, no equilibration- 423 routine freezing. 424 425 In Fertility trial 2, the overall ovulation rate did not differ between the 60 h and 72 h 426 groups (64/65 [98%] vs 40/41 [98%], respectively), but a greater proportion of cows had 427 ovulated at the time of insemination in the 72 h group than in the 60 h group (16/41 [39%] vs 428 6/65 [9%]; P ≤ 0.05). A 2 × 2 interaction effect of insemination timing and semen group on 429 pregnancy rate was significant (P < 0.05; Table 9). Plant-CCTG modified (NE-RF) with 60 h 430 insemination timing had the lowest pregnancy rate compared to other groups (P ≤ 0.05). There 431 was no difference in pregnancy rate between semen extender groups when insemination was 432 done at 72 h. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 22 434 Table 9. Pregnancy rate (number pregnant/number inseminated) in beef cattle following fixed- 435 time artificial insemination with modified semen (NE-RF) in TEYG or plant-CCTG extenders at 436 60 h vs 72 h after progesterone device removal (Fertility trial 2). Semen processing methodTiming of insemination TEYG modified (NE-RF) Plant-CCTG modified (NE-RF) Total 60 h 18/32 (56%)a 4/33 (12%)b 22/65 (34%) 72 h 13/20 (65%)a 12/21 (57%)a 25/41 (61%) Total 31/52 (60%) 16/54 (30%) 47/106 (44%) 437 abWithin rows, values with different superscripts are different (P ≤ 0.05). 438 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris- 439 glycerol extender; NE-RF, no equilibration-modified freezing method. 440 441 Discussion 442 In the present study, the effects of semen processing during cryopreservation were 443 examined by altering equilibration time and freezing rate in different semen extenders. Results 444 demonstrated that beef and bison semen can be frozen without equilibration in a cholesterol- 445 based (animal product-free) extender using a fast freezing method (no equilibration-fast freezing, 446 NE-FF) in 14 min, with acceptable post-thaw sperm motility, plasma membrane integrity, 447 normal acrosomes and in vitro blastocyst formation. In contrast, semen diluted in TEYG 448 extender, directly cooled to 4°C without equilibration and frozen with a fast freezing method 449 (NE-FF) yielded the lowest post-thaw semen quality. Surprisingly, semen diluted in TEYG 450 extender and frozen with a modified method (no equilibration-routine freezing, NE-RF) yielded 451 comparable post-thaw sperm motility and in vivo fertility (up to 65%). .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 23 452 There is no clear consensus in the literature on equilibration time for bull semen. The 453 extended semen is routinely equilibrated for 90-min at 4°C to allow the low density lipoproteins 454 in egg yolk to interact with sperm plasma membranes [36]. These lipoproteins protect sperm 455 from cold shock and lipid-phase transitions that occurs around 15°C during cooling to 4°C 456 [2,16], and replenish phospholipids lost during cryopreservation [37]. In contrast, bull and bison 457 semen diluted in animal- or plant-CCTG was successfully frozen with or without a 90-min 458 equilibration period in the current study. Earlier, we reported that exogenous cholesterol (plant- 459 origin) extender can be used to cryopreserve cattle and bison semen [11,25]. Cholesterol 460 increases the fluidity of sperm plasma membranes and minimizes membrane lipid phase 461 transitions during initial cooling [38]. Data from Experiment 1 showed no difference in post- 462 thaw sperm motilities between animal- and plant-origin cholesterol as expected, since they have 463 identical structural formulas. Therefore, plant cholesterol was used in subsequent experiments as 464 it represents a biosecure alternative to cholesterol from sheep’s wool. 465 Damage to sperm during cryopreservation is twofold. Above 0°C, sperm plasma 466 membrane undergoes phase transition around 15°C. Below 0°C, intracellular ice formation 467 damages subcellular structures [39]. In the routine (RE-RF) method, the diluted semen was 468 equilibrated at 4°C for at least 90 min and frozen as described in previous studies [11,25,30]; 469 whereas in the fast and modified methods, semen was cooled @ -1°C/min from 22°C to 10°C or 470 4°C in a programmable cell freezer eliminating routinely used 90-min equilibration. In addition, 471 the freezing rate was changed from routine (double-ramp: @ -3°C/min from 4°C to -10°C and @ 472 -40°C/min from -10°C to -80°C) to fast (single ramp: @ -40°C/min from 10°C to -80°C) 473 freezing. In Experiments 1 and 2 on beef and bison semen respectively, the TEYG fast-freezing 474 (NE-FF) group had the lowest post-thaw sperm quality (motility, plasma membrane integrity and .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 24 475 normal acrosomes) compared to other groups (TEYG-routine, plant-CCTG-routine and CCTG- 476 fast). In Experiment 3, the cryopreservation process was modified such that equilibration was 477 omitted, but freezing rate below 0°C remained as per routine (NE-RF). Post-thaw quality of 478 semen in TEYG modified and plant-CCTG modified (NE-RF) groups were similar. Previous 479 work revealed that cooling rate (0.1 or 4.2°C/min) had no effect on post-thaw sperm 480 characteristics or in vivo fertility using egg yolk-based extender in dairy bulls [40]. Therefore, 481 cryodamage associated with fast method was likely due to ice nucleation [26] occurring around - 482 40°C in buffalo bull semen [41]. The freezing rate (-3°C/min; ramp 1) used in routine and 483 modified groups in the present study allowed more gradual cell dehydration and ice nucleation 484 [42]. In Experiments 1 and 2, the freezing rate of -40°C/min in ramp 1 was >10× faster, leading 485 to inadequate cell dehydration and subsequent ice nucleation of intracellular free water and 486 crystal formation. Interestingly, CCTG extender provided protection against fast freezing, 487 presumably by adding exogenous cholesterol to sperm plasma membrane, though the exact 488 protective mechanism of exogenous cholesterol in bull and bison sperm is yet to be determined. 489 In previous reports, post-thaw sperm quality and in vivo fertility varied with the use of different 490 equilibration times (24 to 90 h) at 4°C [16,18,19,43]. Contrary to results of the present study, 491 others found that 0 h equilibration of bull semen yielded low sperm motility compared to 4 h 492 [43]. We anticipated that the equilibration process of semen diluted in egg yolk extender is slow, 493 necessitating a prolonged equilibration time [18]. However, our results demonstrate that the long 494 equilibration time with egg yolk extender is required for successful cryopreservation. In vitro 495 cleavage and blastocyst rates were unaffected due to extender or freezing method in the present 496 study. Prior to in vitro fertilization of cattle oocytes, frozen-thawed semen is routinely washed 497 through a density gradient to select for viable sperm, and equal number of viable sperm are co- .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 25 498 incubated with in vitro matured oocytes [25,34,35]. The lack of difference in cleavage and 499 blastocyst rates may have been a result of high number of viable sperm per fertilization droplet 500 that compensates for fertility-associated sperm defects [44-46]. The compensatory effect of 501 sperm number was minimized when sperm number per fertilization droplet was reduced from 502 300,000 to 30,000, and a difference in blastocyte rate due to treatment was more conspicuous 503 [47]. A conventional in vitro fertility trial may not be a reliable assay of in vivo fertility [25], 504 until the sperm concentration per droplet is reduced. 505 North American bison are a significant wildlife reservoir for zoonotic diseases such as 506 bovine tuberculosis and brucellosis. Concerted efforts are underway to conserve and redistribute 507 disease-free bison genetics among long-isolated populations. To this end, it is important to 508 produce biosecure frozen semen to prevent the spread of disease, and to be able to optimize a 509 cryopreservation process that is applicable in field conditions. Fast freezing comes at a cost of 510 increasing sperm damage, particularly with the use of TEYG extender and with the more 511 sensitive bison semen. Like beef bulls, post-thaw sperm characteristics of bison semen diluted in 512 TEYG extender and frozen with the fast method (NE-FF) were adversely affected compared to 513 other treatment groups. However, overall post-thaw sperm quality characteristics were lower in 514 bison compared to beef bulls. This is in consistent with previous findings that bison sperm are 515 more prone to membrane damage compared to cattle [25]. The rapid technique developed herein 516 may be useful for cryopreservation of bison semen under wild conditions for in vitro fertilization 517 later. 518 Fertility trial 1 revealed that the TEYG routine group had a higher pregnancy rate than 519 the plant-CCTG modified group; however, in Fertility trial 2 pregnancy rates were the same 520 between TEYG and plant-CCTG modified freezing groups when insemination was done at 72 h .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 26 521 after progesterone device removal. The lower pregnancy rate using plant-CCTG modified 522 method (NE-RF) inseminated at 60 h may have been a result of asynchrony between sperm 523 viability/longevity and the timing of ovulation. Cholesterol efflux from the plasma membrane is 524 crucial for sperm capacitation and fertilization [13,48], and oversaturation of the sperm plasma 525 membrane with exogenous cholesterol reduced fertility potential and thus demands longer 526 capacitation time [49]. The pregnancy rate was higher in cows synchronized with PRID-Delta 527 than with CIDR, consistent with previous findings [50], perhaps as a result of higher circulating 528 concentrations of progesterone with PRID than CIDR [51]. To our knowledge, this is the first 529 report documenting optimum field fertility using semen frozen in plant-CCTG (egg yolk-free) 530 extender without equilibration. Further investigations are required by including more cows in the 531 fertility trial. However, 58% in vivo fertility (with matching ovarian synchrony) using a 532 biosecure animal product-free extender and short processing time is comparable to cattle industry 533 standards (50-60%), following fixed-time artificial insemination. 534 Conclusion 535 Cattle and bison semen can be cryopreserved without equilibration at 4°C using plant-CCTG 536 extender to achieve acceptable post-thaw sperm quality and in vivo fertility. Cholesterol in plant- 537 CCTG extender requires less time than egg yolk to provide its cryoprotective effect. However, 538 more time may be required between insemination and ovulation to maximize pregnancy rate. 539 Results support the stated hypothesis that a cholesterol-based semen extender obviates the need 540 of extended equilibration for semen cryopreservation, and serves the purpose of enhanced 541 biosecurity, particularly for beef and bison semen collection and processing under field 542 conditions. It is important to consider the synchronization protocol and timing of artificial 543 insemination to achieve optimum fertility in a fixed-time artificial insemination program. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 27 544 Additional studies are required to refine the freezing method using plant-CCTG extender 545 supplemented with antioxidants and membrane stabilizers to improve post-thaw sperm longevity. 546 Acknowledgements 547 Research was supported by grants from the Agriculture and Agri-Food Canada (Research Grant 548 # AGR-14227), the Natural Sciences and Engineering Research Council of Canada, and the 549 Bison Integrated Genomics (BIG) Project from Genome Canada’s Genomic Applications 550 Partnership Program (GAPP) in partnership with Parks Canada and Agriculture and Agri-Food 551 Canada (Grant # 534839). Student salary support was provided by a grant from MITACS (Grant 552 number: IT31476). We thank Kylie Hutt, Laurence de Araujo, Lianne Price, David Moore, Justin 553 Pifko and Jessie Hellquist for their technical assistance and the staff of the Livestock and Forage 554 Centre of Excellence for animal management and care during the study. 555 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint 28 556 References 557 1. Fiser PS, Fairfull RW, Panich PL. Glycerol equilibration time revisted. Reproduction in Domestic 558 Animals. 1995;31: 141-146. 559 2. Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet JW, Crowe JH. Cold shock damage is 560 due to lipid phase transitions in cell membranes: A demonstration using sperm as a model. J Exp 561 Zool. 1993;265: 432-437. 562 3. 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