{"paper_id":"2b543bf8-3c6f-4cc9-ba4f-220c8fcdf267","body_text":"1\n1 Equilibration-free cryopreservation of beef and bison semen\n2\n3 Short title: Semen cryopreservation without equilibration\n4\n5 Yang S1,2, Rajapaksha K2, Zwiefelhofer E1,#a, Adams GP1 and Anzar M2*\n6\n7 1 Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, \n8 University of Saskatchewan, Saskatoon, Canada\n9\n10 2 Agriculture and Agri-Food Canada, Saskatoon Research and Development Center, Saskatoon, \n11 Canada\n12\n13 #aCurrent Address: STgenetics, 22575 Highway 6 S, Navasota, Texas, United States of America\n14\n15 * Corresponding author\n16 E-mail: muhammad.anzar@agr.gc.ca & muhammadanzar786@gmail.com \n17\n18 These authors contributed equally to this work.\n19\n20 CONFLICT OF INTEREST: The authors have declared that no competing interests exist.\n21\n22\n23\n24\n25\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n2\n26 Abstract\n27 Conventional semen cryopreservation involves equilibration at 4°C and optimum freezing rates. \n28 We hypothesized that a cholesterol-based semen extender obviates the need for equilibration, \n29 minimizing total processing time for semen cryopreservation. Experiments were conducted to \n30 determine the effects of semen extender (egg yolk- or cholesterol-based) and freezing method \n31 (routine or fast) on post-thaw sperm characteristics and fertility of beef and bison semen. In \n32 Experiment 1, beef semen diluted in tris-egg yolk-glycerol (TEYG) or cholesterol-cyclodextrin \n33 tris-glycerol (CCTG) extender underwent routine or fast freezing method. Cholesterol from \n34 animal and plant origins were compared. The routine method included 90-min equilibration at \n35 4°C and routine freezing (RE-RF, total time 97 min) whereas the fast method included no \n36 equilibration and fast freezing (NE-FF, total time 14 min). Post-thaw sperm quality was assessed \n37 by CASA, and in vitro fertilization. Post-thaw sperm motility was not affected by the origin of \n38 cholesterol (animal or plant), but was lowest in the TEYG NE-FF group (24% vs 43-51%, P < \n39 0.05). In vitro cleavage and blastocyst development rates did not differ between RE-RF and NE-\n40 FF groups. In Experiment 2, bison semen was diluted in TEYG or plant-CCTG extender and \n41 frozen as in Experiment 1. Post-thaw sperm motility was lowest in the TEYG NE-FF group \n42 (10% vs 39-51%, P < 0.05). In Experiment 3, beef semen diluted in TEYG or plant-CCTG \n43 extender underwent either a routine (RE-RF) or modified freezing (NE-RF, total time 25 min) \n44 method. Post-thaw sperm characteristics did not differ between extenders but were greater using \n45 routine freezing (RE-RF) compared to the modified method of freezing (NE-RF). Pregnancy \n46 rates were similar between extenders (TEYG vs plant-CCTG) using the modified freezing \n47 method without equilibration and insemination at 72 h after progesterone device removal. In \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n3\n48 conclusion, beef and bison semen diluted in cholesterol-based extender may be cryopreserved \n49 without equilibration. (Words 297/300)\n50 KEY WORDS: semen; equilibration; freezing rate; beef; bison\n51\n52 Introduction\n53 Mammalian semen cryopreservation procedures include dilution in cryoprotective extenders, \n54 cooling to 4°C, and freezing below 0°C. Semen extenders increase both ejaculate volume and \n55 sperm longevity. During initial cooling to 4°C, sperm plasma membranes biochemically interact \n56 with extender constituents, specifically referred to as ‘equilibration’ [1]. Mammalian sperm \n57 plasma membranes undergo lipid-phase transitions between 18° to 14°C, known as ‘cold shock’ \n58 [2-4], characterized by lateral movement of phospholipids which increase membrane \n59 permeability to ions, and damages sperm membranes irreversibly [5,6]. Upon ejaculation, binder \n60 of sperm proteins in seminal plasma causes efflux of phospholipids and cholesterol from plasma \n61 membranes. Egg yolk in extender mitigates the cold shock effect by a mechanism that involves \n62 sequestering binder of sperm proteins in seminal plasma [7,8] and binding with sperm plasma \n63 membranes [9], thus stabilizing sperm against membrane lipid-phase transitions and replacing \n64 phospholipids lost during freezing and thawing [10-13].\n65 After dilution in extender, semen is commonly held at 4°C for 90-120 min to achieve \n66 equilibrium between components in semen extender and sperm plasma membranes. With egg \n67 yolk-glycerol extenders, the equilibration time is commonly ≥90 min before freezing below 0°C \n68 [14]. However, a wide range of equilibration time has been reported in literature; long \n69 equilibration time (>12 h) provided better post-thaw sperm quality than short time [1,15-19]. In a \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n4\n70 typical semen cryopreservation protocol, the prerequisite equilibration step prolongs the total \n71 processing time and negatively impacts the efficiency of commercial semen production centers.\n72 Earlier work demonstrated that exogenous cholesterol is incorporated into sperm plasma \n73 membranes within 15 min of incubation in the presence of egg yolk components, and improved \n74 post-thaw sperm motility in beef and bison bulls [20-22]. However, the use of egg yolk, an \n75 animal product, in semen extender raises biosecurity concerns regarding transmission of \n76 infectious diseases. Moreover, egg yolk has undefined composition and varies from batch to \n77 batch. Cholesterol, commonly from sheep’s wool, is known to modulate membrane fluidity, \n78 increase membrane stability, and minimize lipid-phase transitions during cooling [23]. \n79 Alternatively, cholesterol may be sourced from plants to minimize biosecurity risks associated \n80 with animal products and have a defined composition. Cyclodextrins act as carriers to deliver \n81 cholesterol to sperm plasma membrane [22-24]. In this regard, we developed a novel cholesterol-\n82 based extender as an alternative to conventional egg yolk extender for cryopreservation of beef \n83 and bison semen to mitigate biosecurity risks [11,25].\n84 After equilibration, sperm survival below 0°C depends primarily on minimizing two \n85 physico-chemical effects: intracellular ice crystal formation and high solute concentrations [26]. \n86 If cell freezing occurs at an overly fast rate, incomplete dehydration leads to intracellular ice \n87 formation of residual water, causing physical damage to the cell membrane and organelles. \n88 Conversely, if the freezing rate is too slow, prolonged intracellular dehydration leads to high \n89 solute concentrations (solution effect) and toxicity. Glycerol, a low-molecular weight penetrating \n90 cryoprotectant, readily crosses the plasma membrane, binds with free water and helps in \n91 preventing ice crystal formation, and can be added either before or after cooling [1,25,27,28]. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n5\n92 Therefore, total processing time may be further reduced by implementing a fast freezing rate \n93 below 0°C.\n94 North American wood and plains bison are threatened by genetic isolation and disease, \n95 and concerted efforts are underway to rescue the species by establishing a bison biobank with the \n96 use of germplasm in assisted reproductive technologies [25,29-31]. However, semen collection \n97 from free-roaming bison under wild conditions poses logistical concerns related to \n98 cryopreservation processing time and facilities. There is a need for a simple and quick method of \n99 semen cryopreservation to enable collection and processing under remote field conditions and \n100 during extremely cold months.\n101 In the present study, attempts were made to develop a short cryopreservation protocol for \n102 bovine and bison semen by removing the most time-consuming equilibration step, and by \n103 freezing at a faster rate. We hypothesized that a cholesterol-based semen extender obviates the \n104 need for equilibration minimizing total processing time for semen cryopreservation. The specific \n105 objectives of this study were to determine the effects of semen extender (egg yolk- or \n106 cholesterol-based), origin of cholesterol (animal or plant), and freezing method (routine or fast) \n107 on post-thaw sperm quality and fertility of beef and bison semen.\n108 Materials and Methods\n109 Beef cattle and bison bulls were housed separately (5 km apart) under similar management and \n110 nutrition conditions at the Livestock and Forage Center of Excellence, University of \n111 Saskatchewan, Saskatoon. Animal procedures were conducted in accordance with the Canadian \n112 Council on Animal Care and approved by the University of Saskatchewan Animal Care \n113 Committee (Animal Use Protocol #20100150). In all experiments, semen was collected by \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n6\n114 electroejaculation (Pulsator IV Auto Adjust; Lane Manufacturing Inc., Denver, CO, USA). After \n115 collection, semen was transported to the Cryobiology Laboratory, Westgen Research Suite, \n116 University of Saskatchewan, within 2 h at 32°C. Ejaculates were evaluated by computer-assisted \n117 sperm analyzer (CASA; Sperm Vision 3.0, Minitube Canada, Ingersoll, ON, Canada), as \n118 reported earlier [20,32]. Ejaculates possessing sperm concentration >200×106 sperm/mL and \n119 total motility >60% were pooled to minimize bull-to-bull differences, for further processing. All \n120 chemicals were purchased from Sigma-Aldrich (Oakville, ON, Canada) unless otherwise stated.\n121 Semen extender preparation\n122 Cholesterol-cyclodextrin complex (CC) was prepared as previously described [21]. Solution A \n123 was prepared by dissolving 200 mg cholesterol of animal-origin (sheep wool; Cat# C8667) or \n124 plant-origin (PhytoChol®, Wilshire Technologies, Princeton, NJ, USA; Cat# 57-88-5) in 1 mL \n125 chloroform. Solution B was prepared by dissolving 1 g methyl β-cyclodextrin (Cat# C4555) in 2 \n126 mL methanol. Solution A (0.45 mL) was then added to Solution B (2 mL) and mixed until the \n127 solution became homogenous. The mixture was then poured into a glass petri dish and dried \n128 under a gentle stream of nitrogen gas. The resulting crystals were desiccated overnight and \n129 stored in a glass bottle in desiccator at 22°C until used. On the day of experiment, a working \n130 solution of CC (50 mg/mL) was prepared in tris-citric acid (TCA) buffer containing tris-base \n131 3.03% wt/vol, citric acid monohydrate 1.74% wt/vol, and fructose 1.2% wt/vol (pH 7.1) in Milli-\n132 Q distilled water and used immediately.\n133 Tris-glycerol (TG; 2×) extender was prepared by adding glycerol (14% v/v), gentamycin \n134 sulfate (1 mg/mL), tylosin (200 µg/mL; Tylan Soluble, Elanco, Guelph, ON, Canada), and \n135 lincomycin-spectinomycin (600/1200 µg/mL; Linco-Spectin, Pfizer Animal Health, Kirkland, \n136 QC, Canada) to TCA buffer, and stored at -20°C. TG (2×) extender was added to CC treated \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n7\n137 semen (1:1) to achieve the final concentrations of glycerol (7% v/v), gentamycin sulfate (500 \n138 µg/mL), tylosin (100 µg/mL), and lincomycin-spectinomycin (300/600 µg/mL). The combined \n139 extender will be referred to as ‘CCTG’ hereafter.\n140 A conventional tris-egg yolk-glycerol (TEYG) extender was prepared by adding glycerol \n141 (7%, v/v), egg yolk (20%, v/v), gentamycin sulfate (500 µg/mL), tylosin (100 µg/mL), and \n142 lincomycin-spectinomycin (300/600 µg/mL) in TCA buffer. The final extender was centrifuged \n143 at 12,000× g for 15 min at 4°C. The supernatant was recovered and stored at -20°C. All frozen \n144 media were thawed to 37°C on the day of the experiment.\n145 Experiment 1: Effect of extender, origin of cholesterol and freezing \n146 method on post-thaw sperm quality and in vitro fertility of beef bulls\n147 Experiment 1a (Simmental bulls):\n148 Semen was collected from 5 Simmental bulls on 5 different dates (replicates) to determine the \n149 effects of semen extender (egg yolk- or cholesterol-based), source of cholesterol (animal or \n150 plant) and freezing method (routine or fast) on post-thaw sperm motility, and plasma membrane \n151 and acrosome integrity. Pooled ejaculates were divided into three extender groups: TEYG, \n152 animal-CCTG, or plant-CCTG, as previously described [11,25]. For the TEYG group, semen \n153 was diluted to 50×106 sperm/mL with extender (32°C) and held at 22°C for 15 min. For animal- \n154 and plant-CCTG groups, semen was initially diluted to 100×106 sperm/mL with TCA buffer at \n155 32°C, treated with 1 mg animal- or plant-CCTG/ml semen at 22°C for 15 min, and then diluted \n156 1:1 with TG (2×) to achieve a final sperm concentration of 50×106/mL [25]. The final \n157 concentration of animal- or plant-CC was 0.5 mg/ml semen.\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n8\n158 Each TEYG- and CCTG-diluted batch of semen underwent either a routine (routine \n159 equilibration-routine freezing; RE-RF) or fast freezing (no equilibration-fast freezing; NE-FE) \n160 method (Table 1). In the routine method, 15-ml tubes of extended semen were placed in a 500 \n161 mL glass beaker containing water (22°C) and cooled at 4°C for 90 min (equilibration), then filled \n162 into 0.5 mL straws and frozen using a routine freezing curve (i.e., -3°C/min from 4°C to -10°C, -\n163 40°C/min from -10°C to -80°C) in a programmable cell freezer (ICE-CUBE 14-S; Sy-lab \n164 Version 1.30, Gerate GmbH, Neupurkdersdof, Austria) [11]. In the fast method, diluted semen \n165 was immediately filled into 0.5 mL straws at 22°C, and frozen directly in a programmable cell \n166 freezer without equilibration at 4°C (i.e., -1°C/min from 22°C to 10°C, and -40°C/min from \n167 10°C to -80°C). After reaching -80°C in both freezing methods, semen straws were plunged into \n168 liquid nitrogen, and stored until thawing.\n169 For post-thaw sperm analysis, two straws from each treatment group were thawed at \n170 37°C for 1 min and contents were pooled to minimize straw-to-straw variation. Sperm motility \n171 was determined at 0 and 2 h post-thaw by CASA using bull settings [16,32], and in vitro \n172 fertilization potential of sperm in all treatment groups was determined [25,33,34]. Both CASA \n173 and IVF procedures are described under the section below (Semen assays).\n174 Experiment 1b (Angus bulls):\n175 Semen was collected from 5 Angus bulls on 7 different dates (replicates) and pooled, as \n176 described above. Since the effect of CC origin (animal- or plant-derived) was not significant for \n177 any endpoint in Experiment 1a, subsequent experiments were conducted using plant-CCTG \n178 extender for enhanced biosecurity. The effects of semen extender (egg yolk or plant-CCTG) and \n179 freezing method (routine or fast) on post-thaw sperm motility and structural characteristics \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n9\n180 (intact plasma and acrosome membranes) were evaluated at 0 and 2 h, by CASA and flow \n181 cytometer respectively, as described below (Semen assays).\n182 Table 1. Duration of routine and fast freezing methods for cryopreservation of beef and bison \n183 semen (Experiments 1 & 2).\nStage of cryopreservation Routine method\n(RE-RF)*\nFast method\n(NE-FF)*\nCooling/equilibration\n(time)\n22ºC to 4ºC\n(90 min)\n22°C to 10°C @ -1°C/min\n(12 min)\nFreezing ramp 1\n(time)\n4ºC to -10ºC @ -3°C/min\n(~5 min)\n10°C to -80°C @ -40°C/min\n(~2 min)\nFreezing ramp 2\n(time)\n-10ºC to -80ºC @ -40°C/min\n(~2 min) -\nTotal processing time ~97 min ~14 min\n184 *RE-RF: routine equilibration-routine freezing; NE-FF: no equilibration-fast freezing.\n185\n186 Experiment 2: Effect of extender and freezing method on post-thaw \n187 sperm quality of bison bulls\n188 Semen was collected from 5 bison bulls on 5 different dates and pooled, as described above. \n189 Pooled semen was diluted in either TEYG or plant-CCTG extender and underwent routine or fast \n190 method (Table 1). As in Experiment 1, post-thaw sperm motility, and plasma membrane and \n191 normal acrosome were assessed by CASA and flow cytometry respectively, at 0 and 2 h, as \n192 described below (Semen assays). \n193\n194 Experiment 3: Effect of extender and modified freezing method on \n195 post-thaw sperm quality and in vivo fertility of beef bulls\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n10\n196 Semen was collected from 6 Angus bulls on 10 different dates (replicates) and pooled, as \n197 described above. Pooled semen was diluted in either TEYG or plant-CCTG extender and frozen \n198 using either a routine (RE-RF) or modified (no equilibration-routine freezing, NE-RF) method \n199 (Table 2). Semen straws were deep-frozen using a routine freezing curve, as described in \n200 Experiment 1. In the modified group, the extended semen was loaded immediately into 0.5 mL \n201 straws at 22°C and cooled from 22°C to 4°C @ -1°C/min  using a programmable cell freezer \n202 (without equilibration). Below 0°C, the routine freezing curve was used, as described above. \n203 Post-thaw sperm motility, and plasma membrane integrity and normal acrosomes were \n204 determined using CASA and flow cytometry respectively, at 0 and 2 h, as described below \n205 (Semen assays). This experiment was repeated on ten pooled ejaculates (replicates) from four \n206 Angus bulls, on different dates.\n207 Table 2. Duration of routine and modified freezing methods of for cryopreservation of beef \n208 semen (Experiment 3).\nStage of cryopreservation\nRoutine method\n(RE-RF)*\nModified method\n(NE-RF)*\nCooling\n(time)\n22ºC to 4ºC\n(90 min)\n22ºC to 4°C @ -1°C/min\n(18 min)\nFreezing ramp 1\n(time)\n4ºC to -10ºC @ -3°C/min\n(~5 min)\n4ºC to -10ºC @ -3°C/min\n(~5 min)\nFreezing ramp 2\n(time)\n-10ºC to -80ºC @ -40°C/min\n(~2 min)\n-10ºC to -80ºC @ -40°C/min\n(~2 min)\nTotal processing time ~97 min ~25 min\n209 *RE-RF: routine equilibration-routine freezing; NE-RF: no equilibration-routine freezing.\n210\n211\n212\n213\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n11\n214 Fertility trial 1\n215 A fertility trial was conducted to compare the effect of routine semen processing (TEYG \n216 extender and routine freezing [RE-RF]) vs modified semen processing (plant-CCTG extender, \n217 modified freezing [NE-RF]) on pregnancy rate after fixed-time insemination. In addition, two \n218 different intravaginal progesterone devices (CIDR [Zoetis Canada Inc., Kirkland, QC, Canada] \n219 or PRID-Delta [Ceva Animal Health, Cambridge, ON, Canada]) were compared as part of a \n220 separate study. Lactating Angus-cross cows (n = 206) at random stages of the estrous cycle and \n221 at least 40 days post-partum were divided into 3 replicates and synchronized using a standard 5-\n222 day progesterone-based protocol with either a CIDR (n = 105) or PRID-Delta (n = 101; Fig. 1). \n223 On Day 0, cows were given GnRH (100 µg gonadorelin im, Fertilin) and an intravaginal \n224 progesterone device. The progesterone device was removed on Day 5, and cows were given a \n225 luteolytic dose of PGF2α (500 µg cloprostenol im, Bioestrovet; Vetoquinol, Lavaltrie, QC, \n226 Canada) at the time of device removal and again 24 h later. On Day 8 (72 h after PGF2α \n227 treatment), cows were treated with GnRH (100 µg gonadorelin im, Fertilin) and inseminated with \n228 a single dose of TEYG routine (n = 104) or plant-CCTG modified (n = 102) semen, randomly \n229 distributed between synchronization treatments. Ovulation was confirmed by transrectal \n230 ultrasonography at the time of insemination or subsequent examinations 24 and 48 h post-\n231 insemination (MyLab Five, Esaote North America Inc, Fishers, IN, USA), defined as the \n232 disappearance of a large follicle between successive examinations or detection of a new CL. \n233 Pregnancy was diagnosed by transrectal ultrasonography at 27 to 33 days after insemination. The \n234 pregnancy rate was calculated as number pregnant out of those inseminated.\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n12\n235\n236 Fig 1. Fixed-time artificial insemination synchronization protocol. Cows were treated with \n237 GnRH at the time of placement of a progesterone-releasing intravaginal device (CIDR, n = 105 \n238 or PRID-Delta, n = 101). A luteolytic dose of PGF2α was given at the time of device removal \n239 and again 24 h later. Cows were treated with GnRH at the time of insemination 72 h after device \n240 removal using semen processed in 2 different ways (TEYG routine [RE-RF], n = 104 or plant-\n241 CCTG modified [NE-RF], n = 102; Fertility trial 1).\n242 Abbreviations: AI, Artificial insemination; CIDR, Controlled internal drug release; GnRH, \n243 Gonadotropin-releasing hormone; PGF, Prostaglandin F2α; PRID, Progesterone-releasing \n244 intravaginal device; US, Ultrasound.\n245\n246 Fertility trial 2\n247 A follow-up 2×2 fertility trial was conducted to determine the effect of modified semen (no \n248 equilibration-routine freezing, NE-RF) extended in either TEYG or plant-CCTG extenders, and \n249 insemination time (60 h or 72 h after prostaglandin treatment) on pregnancy rate after fixed-time \n250 artificial insemination. Lactating multiparous Hereford-cross cows (n = 106) at random stages of \n251 the estrous cycle and at least 45 days post-partum, were synchronized (Fig. 2). A progesterone-\n252 releasing intravaginal device (CIDR, Vetoquinol, Lavaltrie, QC, Canada) was inserted on Day 0 \n253 and removed on Day 5. A luteolytic dose of PGF2α (500 µg cloprostenol im, Estroplan, \n254 Vetoquinol, Lavaltrie, QC, Canada) was given at the time of device removal. Cows were then \n255 assigned randomly to be inseminated at either 60 h (n = 65) or 72 h (n = 41) after PGF2α \n256 treatment with either TEYG modified (n = 52) or plant-CCTG modified (n = 54) semen group, \n257 and treated with GnRH (100 µg gonadorelin im, Fertilin, Vetoquinol, Lavaltrie, QC, Canada) at \n258 the time of insemination. Ovulation status and pregnancy rates were determined as described in \n259 Fertility trial 1. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n13\n260 Fig 2. Fixed-time artificial insemination synchronization protocol. A progesterone-releasing \n261 intravaginal device (PRID) was placed for a period of 5 days and a luteolytic dose of PGF2α was \n262 given at the time of device removal. Cows were treated with GnRH at the time of insemination at \n263 either 60 h (n = 65) or 72 h (n = 41) after device removal, using modified (NE-RF) semen diluted \n264 TEYG (n = 52) or plant-CCTG (n = 54) extenders (Fertility trial 2).\n265 Abbreviations: AI, Artificial insemination; GnRH, Gonadotropin-releasing hormone; PGF, \n266 Prostaglandin F2α; PRID, Progesterone-releasing intravaginal device; US, Ultrasound.\n267\n268 Semen assays\n269 Computer-assisted sperm analysis\n270 In all experiments, a semen sample (2.5 µl) was placed on a prewarmed (37°C) Leja \n271 chamber slide (depth 20 µm; Leja Products B.V. Nieuw-Vennep, Netherlands) and analyzed \n272 using a computer-assisted sperm analyzer (CASA; Sperm Vision1 3.0, Minitube Canada). A \n273 minimum 200 sperm in 7 fields were assessed for total motility (%; all moving sperm) and \n274 progressive motility (%; sperm moving in straight-line, i.e., more than 10 µm radius with a speed \n275 of > 4.5 µm/s).\n276 Flow cytometer analysis\n277 In Experiments 1b, 2 and 3, plasma and acrosome membrane integrity were assessed by \n278 flow cytometer (CyFlow Space, Partec GmbH, Münster, Germany) using propidium iodide (PI; \n279 stock 2.4 mM in water) and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA; stock 1 \n280 mg/mL in PBS) fluorescent markers, as previously described [16]. Briefly, 1×106 sperm from \n281 each treatment group were diluted in 500 µL TCA buffer and incubated with 5 µL PI and 1 µL \n282 FITC-PNA at 22°C in the dark for 20 min. Sperm were fixed by adding 10 µL 10% \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n14\n283 formaldehyde (v/v). Data acquired by FloMax software (version 2.4, Partec GmbH, Münster, \n284 Germany) revealed four different sperm populations based on plasma and acrosome membrane \n285 integrities: i) sperm with intact plasma membrane and intact acrosome (PI-/FITC-PNA-, intact), \n286 ii) sperm with intact plasma membrane and reacted acrosomes (PI-/FITC-PNA+, partially intact), \n287 iii) sperm with compromised plasma membrane and intact acrosomes (PI+/FITC-PNA-, partially \n288 intact), and iv) sperm with compromised plasma membrane and reacted acrosome (PI+/FITC-\n289 PNA+; compromised). Sperm population with an intact plasma membrane and intact acrosome \n290 (PI-/FITC-PNA-; intact) was selected for further statistical analysis. \n291 In all experiments, a stress test was conducted by holding frozen-thawed semen at 37°C \n292 for 2 h and analyzed for sperm motility and/or structural characteristics with CASA and flow \n293 cytometer respectively.\n294 In vitro fertilization\n295 In Experiment 1a, in vitro fertilization potential of frozen-thawed semen extended in \n296 TEYG, animal-CCTG, or plant-CCTG extender, and frozen with either routine or fast method \n297 were determined. Cattle ovaries were collected from a slaughterhouse near Calgary, Alberta and \n298 transported by air to the Cryobiology Laboratory within 8 h, at 22˚C. In vitro maturation, \n299 fertilization and embryo culture were performed, as previously described [25,34,35]. Briefly, \n300 cumulus-oocyte complexes (COC) were aspirated from follicles (3-8 mm) and identified under a \n301 stereomicroscope (10×). COC with uniform cytoplasm containing >3 layers of cumulus cells \n302 were selected for further processing. COC were washed (3×) in maturation medium (5% calf \n303 serum v/v, 0.5 µg/mL FSH, 5 µg/mL LH and 0.05 µg/mL gentamycin in TCM199). \n304 Approximately 20 COC were pipetted into a 100 µL droplet of maturation medium and \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n15\n305 incubated under mineral oil at 38.5°C and 5% CO2 in air, for 22 h. Frozen-thawed semen \n306 samples were centrifuged (2000× g) through Percoll density gradients (45% and 90%) in a 15 \n307 mL conical tube for 15 min and pellet was diluted to 3×106 sperm/mL with Brackett-Oliphant \n308 (BO) fertilization medium [25]. Mature COC were washed (3×) with BO medium containing \n309 10% (w/v) bovine serum albumin (BSA), placed in 100 µL droplets possessing 300,000 sperm, \n310 and incubated at 38.5°C and 5% CO2 in air, for 18 h (Day 0 = day of IVF). After incubation, the \n311 presumptive zygotes were denuded by repeated pipetting while washing (3×) in 100 µL droplets \n312 of Charles Rosenkrans1 + amino acids culture media (CR1aa) + 5% v/v calf serum under mineral \n313 oil at 38.5°C, 5% CO2, 5% O2 and 90% N2. Cleavage and blastocyst rates were evaluated on Day \n314 2 and 8, respectively, and reported based on the number of oocytes submitted to IVF. This trial \n315 was conducted four times on different dates (replicates).\n316 Statistical analysis\n317 Values are expressed as mean±standard error of the mean (SEM) unless otherwise stated. \n318 In Experiment 1, 3 × 2 factorial analysis was used to study the effect of extenders (TEYG, \n319 animal-CCTG and plant-CCTG), freezing methods (routine and fast) and their interactions on the \n320 post-thaw sperm characteristics. In Experiments 2 and 3, factorial analysis (2 × 2) was used to \n321 determine the effect of extenders and freezing methods. If main effects or their interaction were \n322 significant (P ≤ 0.05), means were compared by Tukey’s test. In vitro cleavage and blastocyst \n323 rates, and in vivo pregnancy rates were compared among groups by binomial generalized linear \n324 mixed model analysis of variance. Data analyses were performed using the R Project (R version \n325 3.3.1, R Foundation for Statistical Computing, Vienna, Austria).\n326 Results\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n16\n327 Experiment 1: Effect of extender, origin of cholesterol and freezing \n328 method on post-thaw sperm quality and in vitro fertility of beef bulls\n329 Experiment 1a (Simmental bulls)\n330 Extender (TEYG, animal-CCTG or plant-CCTG) × freezing method (routine or fast) \n331 interaction was significant on post-thaw total and progressive motility at 0 and 2 h (Table 3). The \n332 TEYG NE-FF group had the lowest post-thaw total and progressive motility compared to other \n333 treatment groups. Post thaw motility did not differ between animal- and plant-origin cholesterol.\n334\n335\n336 Table 3. Post-thaw sperm motility of beef semen diluted in TEYG, animal-CCTG or plant-\n337 CCTG extender and frozen with routine or fast freezing method. Each value represents the \n338 mean±SEM of five pooled ejaculates (replicates), from five Simmental bulls (Experiment 1a).\nRoutine method (RE-RF) Fast method (NE-FF)Sperm \ncharacteristics Time\nTEYG Animal-\nCCTG\nPlant-\nCCTG TEYG Animal\n-CCTG\nPlant-\nCCTG\n0 48±5.0a 50±4.7a 52±5.3a 24±3.2b 47±3.3a 43±3.1aTotal motility \n(%) 2 51±2.6a 50±4.8a 46±7.6a 21±2.5b 45±4.7a 40±8.8a\n0 38±3.5a 43±4.9a 44±5.0a 20±3.1b 38±2.3a 36±2.0aProgressive \nmotility (%) 2 40±3.8a 43±4.5a 40±6.8a 14±2.1b 37±4.2a 32±7.3a\n339 abWithin rows, values with different superscripts are different (P < 0.05). \n340 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris-\n341 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-FF, no equilibration-fast \n342 freezing.\n343\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n17\n344 Following in vitro fertilization of cattle oocytes with frozen-thawed sperm, cleavage and \n345 blastocyst rates ranged from 55% to 65% and 23% to 33%, respectively (Table 4). No effect of \n346 extender or freezing method was detected on cleavage or blastocyst rate.\n347\n348 Table 4. Cleavage and blastocyst rates following in vitro fertilization of bovine oocytes with \n349 beef semen diluted in TEYG, animal-CCTG or plant-CCTG extender and frozen with routine or \n350 fast freezing method. Each value represents four replicates conducted on different dates \n351 (Experiment 1a).\nRoutine method (RE-RF) Fast method (NE-FF)IVF \nvariables TEYG Animal-\nCCTG\nPlant-\nCCTG TEYG Animal-\nCCTG\nPlant-\nCCTG\nCleavage \nrate* \n58/96 \n(60%)\n73/114 \n(64%)\n58/105 \n(55%)\n52/83 \n(63%)\n83/134 \n(62%)\n87/133 \n(65%)\nBlastocyst \nrate* \n29/96 \n(30%)\n35/114 \n(31%)\n29/105 \n(28%)\n19/83 \n(23%)\n38/134 \n(28%)\n44/133 \n(33%)\n352 *Cleavage and blastocyst rates were calculated based on the number of oocytes submitted to \n353 IVF. \n354 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris-\n355 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-FF, no equilibration-fast \n356 freezing.\n357\n358 Experiment 1b (Angus bulls)\n359 Extender × freezing method interactions were significant for all post-thaw sperm \n360 characteristics at 0 and 2 h (Table 5). Once again, all post-thaw sperm characteristics were \n361 lowest in the TEYG fast (NE-FF) group than in other groups. Post-thaw sperm total and \n362 progressive motility, and sperm with intact plasma and acrosome membranes were greater (P < \n363 0.05) in the routine (RE-RF) than the fast (NE-FF) method. Within the fast method, post-thaw \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n18\n364 sperm characteristics at 0 h were significantly greater (P < 0.05) in plant-CCTG than TEYG \n365 extender. Within the routine method, differences between TEYG and plant-CCTG, were not \n366 significant for any sperm characteristic.\n367\n368 Table 5. Post-thaw sperm characteristics of beef semen diluted in TEYG or plant-CCTG \n369 extender and frozen with routine or fast method. Each value represents mean±SEM of seven \n370 pooled ejaculates (replicates), from five Angus bulls (Experiment 1b).\nRoutine method (RE-RF) Fast method (NE-FF)Sperm \ncharacteristics\nTime (h)\nTEYG Plant-CCTG TEYG Plant-CCTG\n0 49±2.6a 42±1.9ab 14±2.5c 32±3.4b\nTotal motility (%) 2 29±1.6a 24±2.1a 5±0.4b 4±0.6b\n0 41±3.1a 33±2.4ab 9±2.1c 24±3.0b\nProgressive motility \n(%) 2 14±1.6a 13±2.5a 1±0.1b 1±0.3b\n0 57±2.7a 64±1.6a 23±1.6c 44±3.8bIntact plasma & \nacrosome \nmembrane (%) 2 40±3.3b 52±2.5a 11±0.7d 22±3.5c\n371 abcdWithin rows, values with no common superscript are different (P < 0.05).\n372 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris-\n373 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-FF, no equilibration-fast \n374 freezing.\n375\n376 Experiment 2: Effect of extender and freezing method on post-thaw sperm \n377 quality of bison bulls \n378 Bison sperm in the TEYG NE-FF group also had the lowest (P < 0.05) total and \n379 progressive motility, and sperm with intact plasma and acrosome membrane integrity compared \n380 to other treatment groups at 0 h post-thaw (Table 6). There was no difference in post-thaw sperm \n381 characteristics between TEYG and plant-CCTG in the routine group (RE-RF), at 0 and 2 h. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n19\n382 Sperm extended in TEYG or plant-CCTG extenders, and underwent the fast method (NE-FF) \n383 yielded lower sperm motility and sperm with intact plasma membrane integrity and normal \n384 acrosomes at 2 h, compared to routine group (RE-RF, P < 0.05).\n385 Table 6. Post-thaw sperm characteristics of bison semen diluted in TEYG or plant-CCTG \n386 extender and frozen with a routine or fast method. Each value represents mean±SEM of five \n387 pooled ejaculates (replicates), from five bison bulls (Experiment 2).\nRoutine method (RE-RF) Fast method (NE-FF)\nSperm characteristics Time (h)\nTEYG Plant-CCTG TEYG Plant-CCTG\n0 45±1.0a 51±4.1a 10±1.2b 39±4.8a\nTotal motility (%) 2 32±2.8a 31±2.1a 3±1.0b 3±0.4b\n0 37±1.0a 45±4.4a 5±1.0b 33±4.7a\nProgressive motility \n(%) 2 26±2.0a 24±2.4a 1±0.2b 1±0.4b\n0 47±1.5a 52±6.1a 17±1.9c 30±4.6bIntact plasma \nmembrane & \nacrosome (%) 2 39±1.9a 42±4.7a 15±1.7b 15±2.8b\n388 abcWithin rows, values with no common superscript are different (P < 0.05).\n389 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris-\n390 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-FF, no equilibration-fast \n391 freezing.\n392\n393 Experiment 3: Effect of extender and modified freezing method on post-thaw \n394 sperm quality and in vivo fertility of beef bulls \n395 There was no effect of extender or interaction between main effects on post-thaw sperm \n396 characteristics. However, sperm motility, and membrane and acrosome integrity were lower in \n397 the modified than routine freezing method (P < 0.05; Table 7).\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n20\n399 Table 7. Post-thaw sperm motility, and plasma membrane integrity and normal acrosomes of \n400 beef semen diluted in TEYG or plant-CCTG and frozen using a routine or modified method. \n401 Each value represents the mean±SEM of ten pooled ejaculates (replicates) from six Angus bulls \n402 (Experiment 3). \nRoutine method \n(RE-RF)\nModified method\n(NE-RF)\nSperm \ncharacteristics\nTime \n(h)\nTEYG Plant-CCTG TEYG Plant-CCTG\n0 45±3.4a 43±1.6a 37±3.4b 37±3.4b\nTotal motility \n(%) 2 33±3.8a 31±2.0a 25±4.0b 22±3.2b\n0 35±3.6a 34±2.2a 26±3.5b 27±3.5b\nProgressive \nmotility (%) 2 23±3.9a 21±2.2a 16±3.1b 12±3.4b\n0 68±2.4a 64±3.4a 56±8.6 b 56±3.9 b\nIntact \nmembranes (%) 2 56±3.6a 52±4.4a 38±9.5b 37±4.8b\n403 abWithin rows, values with different superscripts are different (P < 0.05).\n404 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris-\n405 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-RF, no equilibration-\n406 routine freezing.\n407\n408 In Fertility trial 1, no difference was detected between CIDR and PRID-Delta groups in \n409 ovulation rate (102/105 [97%] vs 101/101 [100%] or interval to ovulation from GnRH treatment \n410 (30.8±1.5 h vs 32.3±1.6 h). Pregnancy rate was greater with PRID-Delta (74/101 [73%] \n411 compared to CIDR (63/105 [60%], P ≤ 0.05), and TEYG routine (RE-RF; 78/104 [75%]) \n412 compared to plant-CCTG modified (NE-RF; 59/102 [58%]; Table 8).\n413\n414\n415\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n21\n416 Table 8. Pregnancy rate (number pregnant/number inseminated) in beef cattle following fixed-\n417 time artificial insemination using CIDR vs PRID-Delta as a source of progesterone for ovarian \n418 synchronization and using either a routine (TEYG, RE-RF) or modified (plant-CCTG, NE-RF) \n419 semen processing method (Fertility trial 1).\nSemen processing methodSynchronization\nprotocol TEYG routine \n(RE-RF)\nPlant-CCTG modified \n(NE-RF)\nTotal\nCIDR 37/52 (71%)a 26/53 (49%)b 63/105 (60%)x\nPRID-Delta 41/52 (79%)a 33/49 (67%)a 74/101 (73%)y\nTotal 78/104 (75%)a 59/102 (58%)b 137/206 (66%)\n420 Values with different superscripts within a row (a,b) and column (x,y) are different (P < 0.05).\n421 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris-\n422 glycerol extender; RE-RF, routine equilibration-routine freezing; NE-RF, no equilibration-\n423 routine freezing.\n424\n425 In Fertility trial 2, the overall ovulation rate did not differ between the 60 h and 72 h \n426 groups (64/65 [98%] vs 40/41 [98%], respectively), but a greater proportion of cows had \n427 ovulated at the time of insemination in the 72 h group than in the 60 h group (16/41 [39%] vs \n428 6/65 [9%]; P ≤ 0.05). A 2 × 2 interaction effect of insemination timing and semen group on \n429 pregnancy rate was significant (P < 0.05; Table 9). Plant-CCTG modified (NE-RF) with 60 h \n430 insemination timing had the lowest pregnancy rate compared to other groups (P ≤ 0.05). There \n431 was no difference in pregnancy rate between semen extender groups when insemination was \n432 done at 72 h.\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n22\n434 Table 9. Pregnancy rate (number pregnant/number inseminated) in beef cattle following fixed-\n435 time artificial insemination with modified semen (NE-RF) in TEYG or plant-CCTG extenders at \n436 60 h vs 72 h after progesterone device removal (Fertility trial 2).\nSemen processing methodTiming of \ninsemination TEYG modified \n(NE-RF)\nPlant-CCTG modified \n(NE-RF)\nTotal\n60 h 18/32 (56%)a 4/33 (12%)b 22/65 (34%)\n72 h 13/20 (65%)a 12/21 (57%)a 25/41 (61%)\nTotal 31/52 (60%) 16/54 (30%) 47/106 (44%)\n437 abWithin rows, values with different superscripts are different (P ≤ 0.05).\n438 Abbreviations: TEYG, tris-egg yolk-glycerol extender; CCTG, cholesterol-cyclodextrin tris-\n439 glycerol extender; NE-RF, no equilibration-modified freezing method.\n440\n441 Discussion \n442 In the present study, the effects of semen processing during cryopreservation were \n443 examined by altering equilibration time and freezing rate in different semen extenders. Results \n444 demonstrated that beef and bison semen can be frozen without equilibration in a cholesterol-\n445 based (animal product-free) extender using a fast freezing method (no equilibration-fast freezing, \n446 NE-FF) in 14 min, with acceptable post-thaw sperm motility, plasma membrane integrity, \n447 normal acrosomes and in vitro blastocyst formation. In contrast, semen diluted in TEYG \n448 extender, directly cooled to 4°C without equilibration and frozen with a fast freezing method \n449 (NE-FF) yielded the lowest post-thaw semen quality. Surprisingly, semen diluted in TEYG \n450 extender and frozen with a modified method (no equilibration-routine freezing, NE-RF) yielded \n451 comparable post-thaw sperm motility and in vivo fertility (up to 65%).\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n23\n452 There is no clear consensus in the literature on equilibration time for bull semen. The \n453 extended semen is routinely equilibrated for 90-min at 4°C to allow the low density lipoproteins \n454 in egg yolk to interact with sperm plasma membranes [36]. These lipoproteins protect sperm \n455 from cold shock and lipid-phase transitions that occurs around 15°C during cooling to 4°C \n456 [2,16], and replenish phospholipids lost during cryopreservation [37]. In contrast, bull and bison \n457 semen diluted in animal- or plant-CCTG was successfully frozen with or without a 90-min \n458 equilibration period in the current study. Earlier, we reported that exogenous cholesterol (plant-\n459 origin) extender can be used to cryopreserve cattle and bison semen [11,25]. Cholesterol \n460 increases the fluidity of sperm plasma membranes and minimizes membrane lipid phase \n461 transitions during initial cooling [38]. Data from Experiment 1 showed no difference in post-\n462 thaw sperm motilities between animal- and plant-origin cholesterol as expected, since they have \n463 identical structural formulas. Therefore, plant cholesterol was used in subsequent experiments as \n464 it represents a biosecure alternative to cholesterol from sheep’s wool.\n465 Damage to sperm during cryopreservation is twofold. Above 0°C, sperm plasma \n466 membrane undergoes phase transition around 15°C. Below 0°C, intracellular ice formation \n467 damages subcellular structures [39]. In the routine (RE-RF) method, the diluted semen was \n468 equilibrated at 4°C for at least 90 min and frozen as described in previous studies [11,25,30]; \n469 whereas in the fast and modified methods, semen was cooled @ -1°C/min from 22°C to 10°C or \n470 4°C in a programmable cell freezer eliminating routinely used 90-min equilibration. In addition, \n471 the freezing rate was changed from routine (double-ramp: @ -3°C/min from 4°C to -10°C and @ \n472 -40°C/min from -10°C to -80°C) to fast (single ramp: @ -40°C/min from 10°C to -80°C) \n473 freezing. In Experiments 1 and 2 on beef and bison semen respectively, the TEYG fast-freezing \n474 (NE-FF) group had the lowest post-thaw sperm quality (motility, plasma membrane integrity and \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n24\n475 normal acrosomes) compared to other groups (TEYG-routine, plant-CCTG-routine and CCTG-\n476 fast). In Experiment 3, the cryopreservation process was modified such that equilibration was \n477 omitted, but freezing rate below 0°C remained as per routine (NE-RF). Post-thaw quality of \n478 semen in TEYG modified and plant-CCTG modified (NE-RF) groups were similar. Previous \n479 work revealed that cooling rate (0.1 or 4.2°C/min) had no effect on post-thaw sperm \n480 characteristics or in vivo fertility using egg yolk-based extender in dairy bulls [40]. Therefore, \n481 cryodamage associated with fast method was likely due to ice nucleation [26] occurring around -\n482 40°C in buffalo bull semen [41]. The freezing rate (-3°C/min; ramp 1) used in routine and \n483 modified groups in the present study allowed more gradual cell dehydration and ice nucleation \n484 [42]. In Experiments 1 and 2, the freezing rate of -40°C/min in ramp 1 was >10× faster, leading \n485 to inadequate cell dehydration and subsequent ice nucleation of intracellular free water and \n486 crystal formation. Interestingly, CCTG extender provided protection against fast freezing, \n487 presumably by adding exogenous cholesterol to sperm plasma membrane, though the exact \n488 protective mechanism of exogenous cholesterol in bull and bison sperm is yet to be determined. \n489 In previous reports, post-thaw sperm quality and in vivo fertility varied with the use of different \n490 equilibration times (24 to 90 h) at 4°C [16,18,19,43]. Contrary to results of the present study, \n491 others found that 0 h equilibration of bull semen yielded low sperm motility compared to 4 h \n492 [43]. We anticipated that the equilibration process of semen diluted in egg yolk extender is slow, \n493 necessitating a prolonged equilibration time [18]. However, our results demonstrate that the long \n494 equilibration time with egg yolk extender is required for successful cryopreservation. In vitro \n495 cleavage and blastocyst rates were unaffected due to extender or freezing method in the present \n496 study. Prior to in vitro fertilization of cattle oocytes, frozen-thawed semen is routinely washed \n497 through a density gradient to select for viable sperm, and equal number of viable sperm are co-\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n25\n498 incubated with in vitro matured oocytes [25,34,35]. The lack of difference in cleavage and \n499 blastocyst rates may have been a result of high number of viable sperm per fertilization droplet \n500 that compensates for fertility-associated sperm defects [44-46]. The compensatory effect of \n501 sperm number was minimized when sperm number per fertilization droplet was reduced from \n502 300,000 to 30,000, and a difference in blastocyte rate due to treatment was more conspicuous \n503 [47]. A conventional in vitro fertility trial may not be a reliable assay of in vivo fertility [25], \n504 until the sperm concentration per droplet is reduced.\n505 North American bison are a significant wildlife reservoir for zoonotic diseases such as \n506 bovine tuberculosis and brucellosis. Concerted efforts are underway to conserve and redistribute \n507 disease-free bison genetics among long-isolated populations. To this end, it is important to \n508 produce biosecure frozen semen to prevent the spread of disease, and to be able to optimize a \n509 cryopreservation process that is applicable in field conditions. Fast freezing comes at a cost of \n510 increasing sperm damage, particularly with the use of TEYG extender and with the more \n511 sensitive bison semen. Like beef bulls, post-thaw sperm characteristics of bison semen diluted in \n512 TEYG extender and frozen with the fast method (NE-FF) were adversely affected compared to \n513 other treatment groups. However, overall post-thaw sperm quality characteristics were lower in \n514 bison compared to beef bulls. This is in consistent with previous findings that bison sperm are \n515 more prone to membrane damage compared to cattle [25]. The rapid technique developed herein \n516 may be useful for cryopreservation of bison semen under wild conditions for in vitro fertilization \n517 later.\n518 Fertility trial 1 revealed that the TEYG routine group had a higher pregnancy rate than \n519 the plant-CCTG modified group; however, in Fertility trial 2 pregnancy rates were the same \n520 between TEYG and plant-CCTG modified freezing groups when insemination was done at 72 h \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n26\n521 after progesterone device removal. The lower pregnancy rate using plant-CCTG modified \n522 method (NE-RF) inseminated at 60 h may have been a result of asynchrony between sperm \n523 viability/longevity and the timing of ovulation. Cholesterol efflux from the plasma membrane is \n524 crucial for sperm capacitation and fertilization [13,48], and oversaturation of the sperm plasma \n525 membrane with exogenous cholesterol reduced fertility potential and thus demands longer \n526 capacitation time [49]. The pregnancy rate was higher in cows synchronized with PRID-Delta \n527 than with CIDR, consistent with previous findings [50], perhaps as a result of higher circulating \n528 concentrations of progesterone with PRID than CIDR [51]. To our knowledge, this is the first \n529 report documenting optimum field fertility using semen frozen in plant-CCTG (egg yolk-free) \n530 extender without equilibration. Further investigations are required by including more cows in the \n531 fertility trial. However, 58% in vivo fertility (with matching ovarian synchrony) using a \n532 biosecure animal product-free extender and short processing time is comparable to cattle industry \n533 standards (50-60%), following fixed-time artificial insemination.\n534 Conclusion \n535 Cattle and bison semen can be cryopreserved without equilibration at 4°C using plant-CCTG \n536 extender to achieve acceptable post-thaw sperm quality and in vivo fertility. Cholesterol in plant-\n537 CCTG extender requires less time than egg yolk to provide its cryoprotective effect. However, \n538 more time may be required between insemination and ovulation to maximize pregnancy rate. \n539 Results support the stated hypothesis that a cholesterol-based semen extender obviates the need \n540 of extended equilibration for semen cryopreservation, and serves the purpose of enhanced \n541 biosecurity, particularly for beef and bison semen collection and processing under field \n542 conditions. It is important to consider the synchronization protocol and timing of artificial \n543 insemination to achieve optimum fertility in a fixed-time artificial insemination program. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n27\n544 Additional studies are required to refine the freezing method using plant-CCTG extender \n545 supplemented with antioxidants and membrane stabilizers to improve post-thaw sperm longevity.\n546 Acknowledgements\n547 Research was supported by grants from the Agriculture and Agri-Food Canada (Research Grant \n548 # AGR-14227), the Natural Sciences and Engineering Research Council of Canada, and the \n549 Bison Integrated Genomics (BIG) Project from Genome Canada’s Genomic Applications \n550 Partnership Program (GAPP) in partnership with Parks Canada and Agriculture and Agri-Food \n551 Canada (Grant # 534839). Student salary support was provided by a grant from MITACS (Grant \n552 number: IT31476). 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It is made \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 16, 2026. ; https://doi.org/10.64898/2026.05.15.725595doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}