Accepted Manuscript Use of commercial extenders and alternatives to prevent sperm agglutination for cryopreservation of brown bear semen S. Gomes-Alves , M. Alvarez , M. Nicolas , E. Lopez -Urueña , C. MartínezRodríguez , S. Borragan , P. de Paz , L. Anel PII:
S0093-691X(14)00237-4
DOI:
10.1016/j.theriogenology.2014.05.015
Reference:
THE 12807
To appear in:
Theriogenology
Received Date: 6 November 2013 Revised Date:
9 May 2014
Accepted Date: 12 May 2014
Please cite this article as: Gomes-Alves S, Alvarez M, Nicolas M, Lopez -Urueña E, MartínezRodríguez C, Borragan S, de Paz P, Anel L, Use of commercial extenders and alternatives to prevent sperm agglutination for cryopreservation of brown bear semen, Theriogenology (2014), doi: 10.1016/ j.theriogenology.2014.05.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised
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Use of commercial extenders and alternatives to prevent sperm agglutination for
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cryopreservation of brown bear semen
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S. Gomes-Alves 1,2, M. Alvarez 1,2, M. Nicolas 1,2, E. Lopez -Urueña1,2, C. Martínez-
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Rodríguez 1,4, S. Borragan3, P. de Paz1,4 and L. Anel 1,2
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ITRA-ULE, INDEGSAL, University of León, 24071, León, Spain.
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Animal Reproduction and Obstetrics, University of León, 24071, León, Spain.
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Cabárceno Park, Cantabria, Spain.
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Molecular Biology, University of León, 24071 León, Spain.
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CORRESPONDING AUTHOR:
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*
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Telephone: +0034987291320
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e-mail: [email protected]
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Department of Molecular Biology, University of León, 24071 León, Spain.
Fax: +0034987291322
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ACCEPTED MANUSCRIPT Abstract
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The objective of this study was to evaluate different bovine and canine commercial
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semen extenders for cryopreservation of brown bear ejaculates and the effect of semen
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collection directly into extender on sperm agglutination. Semen samples were obtained
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by electroejaculation from thirteen adults males. In Experiment 1, 11 ejaculates from
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eight bears were used to evaluate Bioxcell® and Andromed® as extenders, whereas in
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Experiment 2, nine ejaculates from six bears were used to evaluate Triladyl canine®,
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CaniPro®, and Extender 2 as extenders; TTF-ULE/bear served as the control extender
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in both experiments. An extender specifically developed for brown bears (Test-Tris-
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Fructose-egg yolk-glycerol, TTF-ULE/bear) served as a control in both experiments.
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After thawing, total and progressive sperm motility, and sperm viability were greater (P
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< 0.05) for TTF-ULE/bear and Andromed® extenders than for Bioxcell® in Experiment
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1, and greater (P < 0.05) for TTF-ULE/bear extender than for Triladyl Canine®,
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CaniPro®, and Extender 2 in Experiment 2. In Experiment 3, addition of handling
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extender (TTF-H) to the semen collection tube for eight ejaculates from seven bears
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resulted in less (P < 0.05) sperm agglutination in fresh samples (score 0.5 ± 0.2 vs 1.8 ±
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0.4 in diluted and control samples, respectively) with no effect on pre-freeze and post-
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thawing semen quality. In conclusion, TTF-ULE/bear is the most suitable extender for
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brown bear semen cryopreservation, but comparable results can be obtained with the
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commercial extender Andromed®. In addition, collection of ejaculates directly in TTF-
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H extender decreases sperm agglutination in fresh samples.
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Keywords: brown bear ejaculates, spermatozoa freezability, cryopreservation extender,
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sperm agglutination.
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1. Introduction
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The Cantabrian brown bear (Ursus arctos) is considered at risk of extinction (Real
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Decreto 439/1990, regulation of the National Catalogue of Endangered Species) and the
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availability to cryopreserve sperm could help preserve biodiversity in this wild species
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[1,2]. The success of semen cryopreservation depends on several factors, including the
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initial quality of the semen sample, cryopreservation protocol, and freezing extenders
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used [3,4]. It is fundamental to develop specific extender and cryopreservation protocols
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adapted to the characteristics of a particular species [5]. Our group has conducted
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studies in brown bear to develop optimal sperm cryopreservation technique, including
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developing a specific semen extender [6], evaluating different freezing and glycerol
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rates [7], timing of glycerol addition [8], and testing different centrifugation protocols
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[9-11]. Although a bear-specific extender has been successfully used for
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cryopreservation of sperm [7], assessing the suitability of commercial extenders
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designed for other species could provide a practical solution for unexpected situations
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when the specific extenders may not be available.
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To optimize semen cryopreservation protocols, other particularities of brown bear
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ejaculates must also be taken into account. Sperm agglutination is a problem that occurs
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frequently with brown bear ejaculates [5,6,12] and this phenomenon can interfere with
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semen assessment and pre-freezing handling, and possibly affect freezability [5]. In
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brown bear, high or very high level of agglutination is observed in 32% of the ejaculates
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[6]. Thus, based on the fact that agglutination may disappear in boar semen after
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diluting in extender [13], dilution during ejaculate collection might be used as a strategy
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to improve the initial quality of seminal samples in brown bears.
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The objective of the present study was to evaluate different bovine and canine
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commercial semen extenders for cryopreservation of brown bear ejaculates. In addition,
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the effect of semen collection directly into extender on sperm agglutination was also
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evaluated.
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2. Materials and Methods
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2.1. Animals and ejaculate collection
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All experiments were performed after obtaining ethical committee approval from the
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Ethical Committee for Experimentation with Animals of León University, Spain (03-
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02/2010) and all procedures were performed in accordance with Spanish Animal
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Protection Regulation RD1201/2005, which conforms to European Union Regulation
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2003/65. This study was performed at the Cabarceno Park (Cantabria, Spain; 43º 21’ N,
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3º 50’ W; altitude: 143 m). The animals were housed in a half-freedom regime and fed
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chicken meat, bread and fruits.
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Ejaculates from thirteen sexually mature male brown bears were obtained by
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electroejaculation during the breeding season (end of April to early July). The males
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were immobilized using 750 mg of zolazepamHCl and tiletamine HCl (Zoletil100®;
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Virbac, Carros, France) and 6 mg of medetomidine (Zalopin®, Orion Pharma Animal
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Health, Finland, 10 mg/mL), concurrently in the same dart. After immobilization, the
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males were weighed and monitored (pulse, oxygen saturation and breathing) using a
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portable veterinary monitor and were transported to a collection room (18-24 ºC). Prior
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to electroejaculation, the prepucial area was shaved and washed with physiological
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collection to prevent urine contamination. Electroejaculation was carried out with a PT
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Electronics® electroejaculator (PT Electronics, Boring, OR, USA) using a transrectal
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probe 320 mm in length and 26 mm in diameter. Electric stimuli were given until
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ejaculation (10 V and 250 mA, on average) [14]. The ejaculates were collected in
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graduated glass tubes protected in a double-walled conservation tube filled with water at
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30 oC. In Experiment 3, each ejaculate was collected in a funnel and a piping system in
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the form of an “inverted Y” distributing the sample into two tubes with approximately
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equal volume. The collection of ejaculate was performed as isolated fractions to prevent
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urine contamination or low sperm concentration. The sample tubes were transferred to
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the laboratory (22 oC) for analysis and processing. Fractions of reduced concentration (
10 µm/s and straightness > 65 to classify a spermatozoon as motile.
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A flow cytometer (Becton Dickinson Systems, San Jose, CA, USA) equipped with
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standard optics and an argon ion laser, tuned at 488 nm, and running at 200 mW was
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used to determine sperm viability, acrosome integrity, and mitochondrial status. A flow
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rate of 200 sperm/s was used and 10,000 sperm were analyzed per sample. Sperm
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viability was evaluated using SYBR-14 and propidium iodide (PI) (LIVE/DEAD Sperm
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Viability Kit; Invitrogen, Barcelona, Spain) using flow cytometer. Semen samples were
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diluted with PBS to 5 × 106 sperm/mL and 300 µL were transferred to a polypropylene
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tube to which 3 µL PI (24 µM) and 1.5 µL SYBR-14 (100 nM) were added. Sperm with
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positive SYBR-14 fluorescence and negative PI fluorescence were considered live.
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Acrosome integrity was evaluated using PNA-FITC and PI in a stock solution in PBS at
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1 mg/mL and 1.5 mM, respectively. Semen samples were diluted in PBS to 1-2 × 106
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sperm/mL) and 300 µL were transferred to a polypropylene tube to which 0.3 µL of
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stock solution was added. The samples were analyzed after 10 min at room temperature.
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Sperm with negative PNA-FITC and PI fluorescence were considered live with intact
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acrosome. Mitochondrial status was evaluated using JC-1 stain (Invitrogen, Barcelona,
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Spain). Semen samples were diluted in PBS to 1-2 × 106 sperm/mL) and 300 µL were
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transferred to a polypropylene tube to which 0.3 µL of stock solution (3 mM JC-1 in
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DMSO) was added. Sperm emitting orange fluorescence were considered to be positive
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(high mitochondrial membrane potential). Stained samples were incubated at 37 oC for
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20 to 30 min prior to evaluation of sperm viability and mitochondrial status, and for 10
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min at room temperature before evaluation of acrosome integrity.
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2.4. Experimental design and statistical analysis
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In Experiment 1, 11 ejaculates from eight bears were used to evaluate Bioxcell® and
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Andromed® as extenders, whereas in Experiment 2, nine ejaculates from six bears were
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used to evaluate Triladyl canine®, CaniPro®, and Extender 2 as extenders; TTF-
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ULE/bear served as the control extender in both experiments. In Experiment 3, eight
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ejaculates from seven bears were used to evaluate the effect of adding 0.5 mL of
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handling extender (TTF-H) to the semen collection tube. In Experiments 1 and 2, semen
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samples were evaluated just prior to freezing (pre-freezing), and within 10 min after
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thawing (post-thawing). In Experiment 3, semen samples were also evaluated within 10
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min after collection (fresh).
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Statistical analysis was conducted using the SASTM v.9.1 package. The effects of
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treatment (extenders in Experiments 1 and 2, collection method in Experiment 3) were
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determined using Mixed Models with ejaculate included in the models as random effect.
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Significant fixed effects were located using multiple comparisons of means with
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Tukey’s contrasts.
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3. Results
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The volume of ejaculates obtained in this study was 2.2 ± 0.2 mL (mean ± SEM). Fresh
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semen samples had an osmotic pressure of 299.3 ± 4.3 mOsm/kg, pH of 8.3 ± 0.1, and
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sperm concentration of 288.5 ± 44.6 x 106 sperm/mL.
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In Experiment 1, greater (P < 0.05) pre-freezing total and progressive motility were
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observed for TTF-ULE/bear extender than for Bioxcell®. After thawing, total and
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sperm progressive motility, and sperm viability were greater (P < 0.05) for TTF-
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ULE/bear and Andromed® extenders than for Bioxcell®. In addition, Bioxcell®
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resulted in lower (P < 0.05) VAP and VCL than Andromed® and less (P < 0.05)
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acrosome-intact sperm than TTF-ULE/bear (Table 1).
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In Experiment 2, pre-freezing VAP was lower (P < 0.05) for TTF-ULE/bear than for
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CaniPro®, but the proportion of live sperm was greater (P < 0.05) for the former than in
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the latter. After thawing, total and sperm progressive motility, and sperm viability were
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greater (P < 0.05) for TTF-ULE/bear extender than for Triladyl Canine®, CaniPro®,
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and Extender 2. In addition, VAP and VCL were also greater (P < 0.05) for TTF-
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ULE/bear than for Triladyl Canine®. Sperm LIN was greater for CaniPro® than for
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Triladyl Canine® (Table 2).
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In Experiment 3, collection of semen directly into TTF handling extender resulted in
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less (P < 0.05) sperm agglutination in fresh samples (score 0.5 ± 0.2 vs 1.8 ± 0.4 in
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diluted and control samples, respectively). However, there were no effects of collection
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method on sperm agglutination score, motility, kinetics characteristics, viability,
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acrosome integrity, and mitochondrial status in pre-freezing and post-thawing samples
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(data not shown).
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4. Discussion
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Both Andromed® and Bioxcell® are soybean-based extenders and free from animal
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compounds (egg yolk or milk), which is becoming a widespread recommendation to
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avoid the risk of disease transmission [16]. Use of TTF-ULE/bear extender
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supplemented with 5% Type A soybean lecithin for cryopreservation of brown bear
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semen resulted in similar sperm motility to that of the same extender supplemented with
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egg yolk, but sperm viability or acrosomal integrity were lower [17]. In the present, use
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of Andromed® resulted in similar semen quality after cryopreservation when compared
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to TTF-ULE/bear (egg yolk-based). Andromed® has been previously used in other
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species with varying results. Some studies in rams, red deer, and gazelle have
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demonstrated that results obtained with Andromed® were to those obtained with egg
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yolk extenders [18-20], whereas others have observed lower post-thaw semen quality
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with Andromed® when compared to egg yolk extenders for cryopreservation of African
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buffalo, dog, and cat semen [21-24]. The differences observed might be due either
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differences in protective effect of soybean lecithin in semen cryopreservation across
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species or to the source of sperm (epididymal or ejaculated).
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Bioxcell® has been used for cryopreservation of bull [25], ram [26,27] and caprine [28]
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semen with results similar to those obtained using egg yolk or milk extenders. In rams,
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Bioxcell® was more effective than Andromed® in preserving the fertilizing potential of
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chilled or frozen ram semen [29]; however, Bioxcell® resulted in fewer motile sperm
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after freezing of red deer [18]. In the present study, Bioxcell® was less effective in
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protecting brown bear sperm from damage induced by cryopreservation than TTF-
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ULE/bear and Andromed® extenders. These results indicate that Bioxcell® is more
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specifically indicated to ruminants and suggest that the unknown compounds present in
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the extender have only minor protective effect on cell structures of brown bear sperm
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during the cryopreservation process.
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Triladyl canine®, CaniPro® and Extender 2 are egg yolk-based diluents used for
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cryopreservation of canine sperm. The results of the present study indicate that these
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extenders were not as effective TTF-ULE/bear extender for cryopreservation of brown
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bear sperm. Although the fractions of CaniPro® and Extender 2 extenders containing
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glycerol are usually only added after cooling, post-thaw sperm motility and viability
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were similar when glycerol was added at room temperature or at 5 ºC to TTF-ULE/bear
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extender in a previous study [8], thus indicating that brown bear sperm tolerant well the
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addition of glycerol at room temperature. All egg yolk-based extenders used in the
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present study have the same concentration of egg yolk and very similar concentration of
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glycerol, suggesting that the differences among extenders are related to other extender
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components.
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Sugars also play an important role in semen extenders, providing energy substrates and
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acting as osmotic and cryoprotective agents [30]. Okano et al. [31] referred to studies
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that demonstrated that efficient sugar supplementation varies across the species.
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However, Japanese black bear semen could be frozen with any of the three examined
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diluents that contained different sugars (glucose, fructose, and fructose-lactose-
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raffinose). Two extenders used in the present study contained fructose (TTF-ULE/bear
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and Andromed®) and two contained glucose (CaniPro® and Extender 2); the other
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extenders were unknown. These observations seem to suggest that fructose is a better
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alternative to glucose for brown bear semen extenders.
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particles present in the sample. Monga and Roberts [32] provided evidence that
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fimbriated bacteria can adhere to the glycoproteins of spermatozoa surface inducing
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agglutination of motile sperm. In addition, specific sperm surface proteins appear to be
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involved in sperm agglutination [33,34]. Collecting brown bear ejaculates directly into
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extender significantly reduced the degree of sperm agglutination in fresh samples
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without affecting pre-freezing and post-thawing semen quality. Reduced agglutination
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might be related to reduction of bacterial activity and/or masking sperm surface
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proteins.
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In conclusion, TTF-ULE/bear is the most suitable extender for brown bear semen
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cryopreservation, but comparable results can be obtained with the commercial extender
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Andromed®. In addition, collection of ejaculates directly in TTF-H extender decreases
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sperm agglutination in fresh samples.
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Acknowledgements
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The authors thank Miguel Angel Marañon, Miguel Angel Prieto and other game keepers
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of Cabárceno Park (Cantabria, Spain), and Maria Mata Campuzano, Manuel Alvarez
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Rodríguez, Patricia Manrique Revuelta, Carmen Martinez Rodriguez and Luis Anel-
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Lopez. This work was supported by MICINN (CGL2010-19213/BOS) and CANTUR
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S.A. Susana Gomes-Alves was supported by a PhD fellowship (SFRH/BD/45522/2008,
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co-founded by POPH – QREN).
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[26] Gil J, Lundeheim N, Soderquist L, Rodriiuez-Martinez H. Influence of extender,
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temperature, and addition of glycerol on post-thaw sperm parameters in ram semen.
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Theriogenology 2003a;59(5-6):1241-55.
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[27] Gil J, Rodriguez-Irazoqui M, Lundeheim N, Soderquist L, Rodriguez-Martinez H.
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Fertility of ram semen frozen in bioexcell and used for cervical artificial insemination.
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Theriogenology 2003b;59(5-6):1157-70.
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[28] Roof DJ, Bowley S, Price LL, Matsas DJ. Comparison of two commercial
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extenders for cryopreservation of goat semen without sperm washing. Theriogenology
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2012;77(2):412-20.
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extenders with field fertility of stored ram (Ovis aries) semen: A randomized double-
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[30] Salamon S, Maxwell WM. Storage of ram semen. Anim. Reprod. Sci. 2000;62(1-
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3):77-111.
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[31] Okano T, Murase T, Yayota C, Komatsu T, Miyazawa K, Asano M, et al. Characteristics of
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captive japanese black bears (Ursus thibetanus japonicus) semen collected by
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electroejaculation with different voltages for stimulation and frozen-thawed under different
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conditions. Anim. Reprod. Sci. 2006;95(1-2):134-43.
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[32] Monga M, Roberts JA. Spermagglutination by bacteria: Receptor-specific
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interactions. J. Androl. 1994;15(2):151-6.
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[33] Fornes MW, Burgos MH. Epididymal glycoprotein involved in rat sperm
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association. Mol. Reprod. Dev. 1994;38(1):43-7.
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[34] Flaherty SP, Swann NJ, Primakoff P, Myles DG. A role for the WH-30 protein in
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sperm-sperm adhesion during rouleaux formation in the guinea pig. Dev. Biol.
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1993;156(1):243-52.
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Linearity (%)
Live (%)
Acrosomeintact (%)
High mitochondria potential (%)
65.2±5.8 79.2±8.3 75.2±7.8
109.2±10.0 135.8±14.8 121.5±13.1
42.7±2.0 37.9±2.4 40.7±2.5
67.1±4.7 67.3±4.8 63.8±5.6
67.6±4.7 68.1±3.8 67.0±4.1
56.3±9.4 57.3±7.9 51.8±9.8
35.0±1.1 35.6±1.0 36.7±2.0
44.4±4.1a 42.1±3.9a 29.4±3.8b
46.1±3.4a 41.1±4.4a 33.3±4.9b
29.1±8.0 38.5±8.0 20.9±4.7
44.3±4.4ab 58.5±5.4a 36.3±5.8b
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Curvilinear velocity (µm/s)
79.8±8.7ab 110.7±11.5a 64.2±10.2b
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Pre-freezing TTF-ULE/bear 74.7±4.3a 47.6±5.1a 39.6±7.0ab Andromed® 63.3±7.6a b Bioxcell® 50.1±7.8 29.3±6.5b Post-thawing 23.8±2.9a TTF-ULE/bear 55.5±6.2a Andromed® 54.5±5.2a 27.9±3.3a b Bioxcell® 17.8±5.3 7.2±2.3b a,b Rows with different superscripts differ (P < 0.05).
Average path velocity (µm/s)
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Progressive motility (%)
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Total motility (%)
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Table 1. Sperm motility, kinetics, viability, acrosome integrity, and mitochondrial status (mean ± SEM) in brown bear semen according to the type of extender (n = 11).
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Linearity (%)
Live (%)
Acrosomeintact (%)
High mitochondria potential (%)
61.0±6.1a 69.5±4.9ab 75.9±6.2b 70.3±5.7ab
116.1±13.7 119.2±11.2 131.8±11.8 122.1±11.0
37.5±2.5 40.9±2.0 39.4±2.0 35.5±1.8
71,8±4,1a 59,2±5,6b 66,8±5,9ab 66,5±5,6ab
76,2±3,2 68,3±5 71,7±5,5 71,1±5,1
29,6±8 40,4±9,7 29,1±7 38,8±8,7
35.0±1.8ab 32.9±1.1b 37.2±1.3a 35.3±1.4ab
47,2±4,6a 31,9±3,6b 32,9±1,4b 33,9±5,2b
50,2±4,9 41,5±3,4 41,8±6,4 41,6±5,4
29,1±10 36,9±5,9 32±5,5 33,8±6,2
52.4±5.2a 30.6±5.3b 46.1±8.1a 42.3±7.6ab
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Curvilinear velocity (µm/s)
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Pre-freezing TTF-ULE/bear 77.6±5.1 46.8±6.8 Triladyl Canine® 72.0±6.8 41.5±4.5 CaniPro® 69.4±5.3 38.3±3.7 Extender 2 75.0±5.5 35.8±6.4 Post-thawing TTF-ULE/bear 71.8±8.2a 35.7±4.3a b 15.7±2.7b Triladyl Canine® 48.6±6.1 CaniPro® 46.3±8.8b 21.4±5.3b b Extender 2 47.9±8.3 20.6±4.5b a,b Rows with different superscripts differ (P < 0.05).
Average path velocity (µm/s)
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98.8±9.8a 53.0±8.3b 83.0±15.4ab 73.7±12.2ab
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Total motility (%)
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Table 2. Sperm motility, kinetics, viability, acrosome integrity, and mitochondrial status (mean ± SEM) in brown bear semen according to the type of extender (n = 9).
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S0093-691X(14)00237-4
DOI:
10.1016/j.theriogenology.2014.05.015
Reference:
THE 12807
To appear in:
Theriogenology
Received Date: 6 November 2013 Revised Date:
9 May 2014
Accepted Date: 12 May 2014
Please cite this article as: Gomes-Alves S, Alvarez M, Nicolas M, Lopez -Urueña E, MartínezRodríguez C, Borragan S, de Paz P, Anel L, Use of commercial extenders and alternatives to prevent sperm agglutination for cryopreservation of brown bear semen, Theriogenology (2014), doi: 10.1016/ j.theriogenology.2014.05.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised
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Use of commercial extenders and alternatives to prevent sperm agglutination for
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cryopreservation of brown bear semen
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S. Gomes-Alves 1,2, M. Alvarez 1,2, M. Nicolas 1,2, E. Lopez -Urueña1,2, C. Martínez-
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Rodríguez 1,4, S. Borragan3, P. de Paz1,4 and L. Anel 1,2
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ITRA-ULE, INDEGSAL, University of León, 24071, León, Spain.
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Animal Reproduction and Obstetrics, University of León, 24071, León, Spain.
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Cabárceno Park, Cantabria, Spain.
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Molecular Biology, University of León, 24071 León, Spain.
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CORRESPONDING AUTHOR:
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*
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Telephone: +0034987291320
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e-mail: [email protected]
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Department of Molecular Biology, University of León, 24071 León, Spain.
Fax: +0034987291322
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ACCEPTED MANUSCRIPT Abstract
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The objective of this study was to evaluate different bovine and canine commercial
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semen extenders for cryopreservation of brown bear ejaculates and the effect of semen
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collection directly into extender on sperm agglutination. Semen samples were obtained
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by electroejaculation from thirteen adults males. In Experiment 1, 11 ejaculates from
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eight bears were used to evaluate Bioxcell® and Andromed® as extenders, whereas in
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Experiment 2, nine ejaculates from six bears were used to evaluate Triladyl canine®,
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CaniPro®, and Extender 2 as extenders; TTF-ULE/bear served as the control extender
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in both experiments. An extender specifically developed for brown bears (Test-Tris-
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Fructose-egg yolk-glycerol, TTF-ULE/bear) served as a control in both experiments.
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After thawing, total and progressive sperm motility, and sperm viability were greater (P
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< 0.05) for TTF-ULE/bear and Andromed® extenders than for Bioxcell® in Experiment
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1, and greater (P < 0.05) for TTF-ULE/bear extender than for Triladyl Canine®,
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CaniPro®, and Extender 2 in Experiment 2. In Experiment 3, addition of handling
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extender (TTF-H) to the semen collection tube for eight ejaculates from seven bears
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resulted in less (P < 0.05) sperm agglutination in fresh samples (score 0.5 ± 0.2 vs 1.8 ±
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0.4 in diluted and control samples, respectively) with no effect on pre-freeze and post-
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thawing semen quality. In conclusion, TTF-ULE/bear is the most suitable extender for
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brown bear semen cryopreservation, but comparable results can be obtained with the
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commercial extender Andromed®. In addition, collection of ejaculates directly in TTF-
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H extender decreases sperm agglutination in fresh samples.
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Keywords: brown bear ejaculates, spermatozoa freezability, cryopreservation extender,
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sperm agglutination.
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1. Introduction
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The Cantabrian brown bear (Ursus arctos) is considered at risk of extinction (Real
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Decreto 439/1990, regulation of the National Catalogue of Endangered Species) and the
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availability to cryopreserve sperm could help preserve biodiversity in this wild species
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[1,2]. The success of semen cryopreservation depends on several factors, including the
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initial quality of the semen sample, cryopreservation protocol, and freezing extenders
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used [3,4]. It is fundamental to develop specific extender and cryopreservation protocols
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adapted to the characteristics of a particular species [5]. Our group has conducted
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studies in brown bear to develop optimal sperm cryopreservation technique, including
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developing a specific semen extender [6], evaluating different freezing and glycerol
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rates [7], timing of glycerol addition [8], and testing different centrifugation protocols
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[9-11]. Although a bear-specific extender has been successfully used for
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cryopreservation of sperm [7], assessing the suitability of commercial extenders
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designed for other species could provide a practical solution for unexpected situations
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when the specific extenders may not be available.
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To optimize semen cryopreservation protocols, other particularities of brown bear
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ejaculates must also be taken into account. Sperm agglutination is a problem that occurs
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frequently with brown bear ejaculates [5,6,12] and this phenomenon can interfere with
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semen assessment and pre-freezing handling, and possibly affect freezability [5]. In
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brown bear, high or very high level of agglutination is observed in 32% of the ejaculates
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[6]. Thus, based on the fact that agglutination may disappear in boar semen after
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diluting in extender [13], dilution during ejaculate collection might be used as a strategy
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to improve the initial quality of seminal samples in brown bears.
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The objective of the present study was to evaluate different bovine and canine
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commercial semen extenders for cryopreservation of brown bear ejaculates. In addition,
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the effect of semen collection directly into extender on sperm agglutination was also
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evaluated.
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2. Materials and Methods
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2.1. Animals and ejaculate collection
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All experiments were performed after obtaining ethical committee approval from the
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Ethical Committee for Experimentation with Animals of León University, Spain (03-
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02/2010) and all procedures were performed in accordance with Spanish Animal
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Protection Regulation RD1201/2005, which conforms to European Union Regulation
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2003/65. This study was performed at the Cabarceno Park (Cantabria, Spain; 43º 21’ N,
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3º 50’ W; altitude: 143 m). The animals were housed in a half-freedom regime and fed
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chicken meat, bread and fruits.
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Ejaculates from thirteen sexually mature male brown bears were obtained by
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electroejaculation during the breeding season (end of April to early July). The males
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were immobilized using 750 mg of zolazepamHCl and tiletamine HCl (Zoletil100®;
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Virbac, Carros, France) and 6 mg of medetomidine (Zalopin®, Orion Pharma Animal
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Health, Finland, 10 mg/mL), concurrently in the same dart. After immobilization, the
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males were weighed and monitored (pulse, oxygen saturation and breathing) using a
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portable veterinary monitor and were transported to a collection room (18-24 ºC). Prior
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to electroejaculation, the prepucial area was shaved and washed with physiological
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collection to prevent urine contamination. Electroejaculation was carried out with a PT
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Electronics® electroejaculator (PT Electronics, Boring, OR, USA) using a transrectal
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probe 320 mm in length and 26 mm in diameter. Electric stimuli were given until
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ejaculation (10 V and 250 mA, on average) [14]. The ejaculates were collected in
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graduated glass tubes protected in a double-walled conservation tube filled with water at
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30 oC. In Experiment 3, each ejaculate was collected in a funnel and a piping system in
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the form of an “inverted Y” distributing the sample into two tubes with approximately
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equal volume. The collection of ejaculate was performed as isolated fractions to prevent
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urine contamination or low sperm concentration. The sample tubes were transferred to
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the laboratory (22 oC) for analysis and processing. Fractions of reduced concentration (
10 µm/s and straightness > 65 to classify a spermatozoon as motile.
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A flow cytometer (Becton Dickinson Systems, San Jose, CA, USA) equipped with
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standard optics and an argon ion laser, tuned at 488 nm, and running at 200 mW was
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used to determine sperm viability, acrosome integrity, and mitochondrial status. A flow
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rate of 200 sperm/s was used and 10,000 sperm were analyzed per sample. Sperm
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viability was evaluated using SYBR-14 and propidium iodide (PI) (LIVE/DEAD Sperm
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Viability Kit; Invitrogen, Barcelona, Spain) using flow cytometer. Semen samples were
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diluted with PBS to 5 × 106 sperm/mL and 300 µL were transferred to a polypropylene
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tube to which 3 µL PI (24 µM) and 1.5 µL SYBR-14 (100 nM) were added. Sperm with
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positive SYBR-14 fluorescence and negative PI fluorescence were considered live.
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Acrosome integrity was evaluated using PNA-FITC and PI in a stock solution in PBS at
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1 mg/mL and 1.5 mM, respectively. Semen samples were diluted in PBS to 1-2 × 106
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sperm/mL) and 300 µL were transferred to a polypropylene tube to which 0.3 µL of
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stock solution was added. The samples were analyzed after 10 min at room temperature.
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Sperm with negative PNA-FITC and PI fluorescence were considered live with intact
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acrosome. Mitochondrial status was evaluated using JC-1 stain (Invitrogen, Barcelona,
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Spain). Semen samples were diluted in PBS to 1-2 × 106 sperm/mL) and 300 µL were
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transferred to a polypropylene tube to which 0.3 µL of stock solution (3 mM JC-1 in
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DMSO) was added. Sperm emitting orange fluorescence were considered to be positive
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(high mitochondrial membrane potential). Stained samples were incubated at 37 oC for
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20 to 30 min prior to evaluation of sperm viability and mitochondrial status, and for 10
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min at room temperature before evaluation of acrosome integrity.
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2.4. Experimental design and statistical analysis
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In Experiment 1, 11 ejaculates from eight bears were used to evaluate Bioxcell® and
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Andromed® as extenders, whereas in Experiment 2, nine ejaculates from six bears were
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used to evaluate Triladyl canine®, CaniPro®, and Extender 2 as extenders; TTF-
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ULE/bear served as the control extender in both experiments. In Experiment 3, eight
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ejaculates from seven bears were used to evaluate the effect of adding 0.5 mL of
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handling extender (TTF-H) to the semen collection tube. In Experiments 1 and 2, semen
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samples were evaluated just prior to freezing (pre-freezing), and within 10 min after
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thawing (post-thawing). In Experiment 3, semen samples were also evaluated within 10
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min after collection (fresh).
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Statistical analysis was conducted using the SASTM v.9.1 package. The effects of
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treatment (extenders in Experiments 1 and 2, collection method in Experiment 3) were
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determined using Mixed Models with ejaculate included in the models as random effect.
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Significant fixed effects were located using multiple comparisons of means with
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Tukey’s contrasts.
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3. Results
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The volume of ejaculates obtained in this study was 2.2 ± 0.2 mL (mean ± SEM). Fresh
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semen samples had an osmotic pressure of 299.3 ± 4.3 mOsm/kg, pH of 8.3 ± 0.1, and
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sperm concentration of 288.5 ± 44.6 x 106 sperm/mL.
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In Experiment 1, greater (P < 0.05) pre-freezing total and progressive motility were
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observed for TTF-ULE/bear extender than for Bioxcell®. After thawing, total and
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sperm progressive motility, and sperm viability were greater (P < 0.05) for TTF-
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ULE/bear and Andromed® extenders than for Bioxcell®. In addition, Bioxcell®
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resulted in lower (P < 0.05) VAP and VCL than Andromed® and less (P < 0.05)
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acrosome-intact sperm than TTF-ULE/bear (Table 1).
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In Experiment 2, pre-freezing VAP was lower (P < 0.05) for TTF-ULE/bear than for
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CaniPro®, but the proportion of live sperm was greater (P < 0.05) for the former than in
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the latter. After thawing, total and sperm progressive motility, and sperm viability were
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greater (P < 0.05) for TTF-ULE/bear extender than for Triladyl Canine®, CaniPro®,
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and Extender 2. In addition, VAP and VCL were also greater (P < 0.05) for TTF-
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ULE/bear than for Triladyl Canine®. Sperm LIN was greater for CaniPro® than for
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Triladyl Canine® (Table 2).
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In Experiment 3, collection of semen directly into TTF handling extender resulted in
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less (P < 0.05) sperm agglutination in fresh samples (score 0.5 ± 0.2 vs 1.8 ± 0.4 in
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diluted and control samples, respectively). However, there were no effects of collection
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method on sperm agglutination score, motility, kinetics characteristics, viability,
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acrosome integrity, and mitochondrial status in pre-freezing and post-thawing samples
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(data not shown).
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4. Discussion
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Both Andromed® and Bioxcell® are soybean-based extenders and free from animal
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compounds (egg yolk or milk), which is becoming a widespread recommendation to
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avoid the risk of disease transmission [16]. Use of TTF-ULE/bear extender
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supplemented with 5% Type A soybean lecithin for cryopreservation of brown bear
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semen resulted in similar sperm motility to that of the same extender supplemented with
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egg yolk, but sperm viability or acrosomal integrity were lower [17]. In the present, use
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of Andromed® resulted in similar semen quality after cryopreservation when compared
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to TTF-ULE/bear (egg yolk-based). Andromed® has been previously used in other
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species with varying results. Some studies in rams, red deer, and gazelle have
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demonstrated that results obtained with Andromed® were to those obtained with egg
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yolk extenders [18-20], whereas others have observed lower post-thaw semen quality
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with Andromed® when compared to egg yolk extenders for cryopreservation of African
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buffalo, dog, and cat semen [21-24]. The differences observed might be due either
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differences in protective effect of soybean lecithin in semen cryopreservation across
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species or to the source of sperm (epididymal or ejaculated).
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Bioxcell® has been used for cryopreservation of bull [25], ram [26,27] and caprine [28]
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semen with results similar to those obtained using egg yolk or milk extenders. In rams,
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Bioxcell® was more effective than Andromed® in preserving the fertilizing potential of
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chilled or frozen ram semen [29]; however, Bioxcell® resulted in fewer motile sperm
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after freezing of red deer [18]. In the present study, Bioxcell® was less effective in
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protecting brown bear sperm from damage induced by cryopreservation than TTF-
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ULE/bear and Andromed® extenders. These results indicate that Bioxcell® is more
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specifically indicated to ruminants and suggest that the unknown compounds present in
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the extender have only minor protective effect on cell structures of brown bear sperm
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during the cryopreservation process.
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Triladyl canine®, CaniPro® and Extender 2 are egg yolk-based diluents used for
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cryopreservation of canine sperm. The results of the present study indicate that these
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extenders were not as effective TTF-ULE/bear extender for cryopreservation of brown
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bear sperm. Although the fractions of CaniPro® and Extender 2 extenders containing
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glycerol are usually only added after cooling, post-thaw sperm motility and viability
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were similar when glycerol was added at room temperature or at 5 ºC to TTF-ULE/bear
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extender in a previous study [8], thus indicating that brown bear sperm tolerant well the
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addition of glycerol at room temperature. All egg yolk-based extenders used in the
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present study have the same concentration of egg yolk and very similar concentration of
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glycerol, suggesting that the differences among extenders are related to other extender
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components.
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Sugars also play an important role in semen extenders, providing energy substrates and
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acting as osmotic and cryoprotective agents [30]. Okano et al. [31] referred to studies
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that demonstrated that efficient sugar supplementation varies across the species.
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However, Japanese black bear semen could be frozen with any of the three examined
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diluents that contained different sugars (glucose, fructose, and fructose-lactose-
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raffinose). Two extenders used in the present study contained fructose (TTF-ULE/bear
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and Andromed®) and two contained glucose (CaniPro® and Extender 2); the other
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extenders were unknown. These observations seem to suggest that fructose is a better
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alternative to glucose for brown bear semen extenders.
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particles present in the sample. Monga and Roberts [32] provided evidence that
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fimbriated bacteria can adhere to the glycoproteins of spermatozoa surface inducing
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agglutination of motile sperm. In addition, specific sperm surface proteins appear to be
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involved in sperm agglutination [33,34]. Collecting brown bear ejaculates directly into
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extender significantly reduced the degree of sperm agglutination in fresh samples
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without affecting pre-freezing and post-thawing semen quality. Reduced agglutination
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might be related to reduction of bacterial activity and/or masking sperm surface
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proteins.
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In conclusion, TTF-ULE/bear is the most suitable extender for brown bear semen
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cryopreservation, but comparable results can be obtained with the commercial extender
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Andromed®. In addition, collection of ejaculates directly in TTF-H extender decreases
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sperm agglutination in fresh samples.
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Acknowledgements
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The authors thank Miguel Angel Marañon, Miguel Angel Prieto and other game keepers
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of Cabárceno Park (Cantabria, Spain), and Maria Mata Campuzano, Manuel Alvarez
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Rodríguez, Patricia Manrique Revuelta, Carmen Martinez Rodriguez and Luis Anel-
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Lopez. This work was supported by MICINN (CGL2010-19213/BOS) and CANTUR
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S.A. Susana Gomes-Alves was supported by a PhD fellowship (SFRH/BD/45522/2008,
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co-founded by POPH – QREN).
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[3] Gilmore JA, McGann LE, Ashworth E, Acker JP, Raath JP, Bush M, et al.
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biodiversity preservation through the development of genome resource banking. Anim.
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[6] Anel L, Gomes-Alves S, Alvarez M, Borragan S, Anel E, Nicolas M, et al. Effect of
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basic factors of extender composition on post-thawing quality of brown bear
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electroejaculated spermatozoa. Theriogenology 2010;74(4):643-51.
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[7] de Paz P, Alvarez-Rodriguez M, Nicolas M, Alvarez M, Chamorro C, Borragan S, et
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ACCEPTED MANUSCRIPT
Linearity (%)
Live (%)
Acrosomeintact (%)
High mitochondria potential (%)
65.2±5.8 79.2±8.3 75.2±7.8
109.2±10.0 135.8±14.8 121.5±13.1
42.7±2.0 37.9±2.4 40.7±2.5
67.1±4.7 67.3±4.8 63.8±5.6
67.6±4.7 68.1±3.8 67.0±4.1
56.3±9.4 57.3±7.9 51.8±9.8
35.0±1.1 35.6±1.0 36.7±2.0
44.4±4.1a 42.1±3.9a 29.4±3.8b
46.1±3.4a 41.1±4.4a 33.3±4.9b
29.1±8.0 38.5±8.0 20.9±4.7
44.3±4.4ab 58.5±5.4a 36.3±5.8b
SC
Curvilinear velocity (µm/s)
79.8±8.7ab 110.7±11.5a 64.2±10.2b
TE D
Pre-freezing TTF-ULE/bear 74.7±4.3a 47.6±5.1a 39.6±7.0ab Andromed® 63.3±7.6a b Bioxcell® 50.1±7.8 29.3±6.5b Post-thawing 23.8±2.9a TTF-ULE/bear 55.5±6.2a Andromed® 54.5±5.2a 27.9±3.3a b Bioxcell® 17.8±5.3 7.2±2.3b a,b Rows with different superscripts differ (P < 0.05).
Average path velocity (µm/s)
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Progressive motility (%)
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Total motility (%)
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Table 1. Sperm motility, kinetics, viability, acrosome integrity, and mitochondrial status (mean ± SEM) in brown bear semen according to the type of extender (n = 11).
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ACCEPTED MANUSCRIPT
Linearity (%)
Live (%)
Acrosomeintact (%)
High mitochondria potential (%)
61.0±6.1a 69.5±4.9ab 75.9±6.2b 70.3±5.7ab
116.1±13.7 119.2±11.2 131.8±11.8 122.1±11.0
37.5±2.5 40.9±2.0 39.4±2.0 35.5±1.8
71,8±4,1a 59,2±5,6b 66,8±5,9ab 66,5±5,6ab
76,2±3,2 68,3±5 71,7±5,5 71,1±5,1
29,6±8 40,4±9,7 29,1±7 38,8±8,7
35.0±1.8ab 32.9±1.1b 37.2±1.3a 35.3±1.4ab
47,2±4,6a 31,9±3,6b 32,9±1,4b 33,9±5,2b
50,2±4,9 41,5±3,4 41,8±6,4 41,6±5,4
29,1±10 36,9±5,9 32±5,5 33,8±6,2
52.4±5.2a 30.6±5.3b 46.1±8.1a 42.3±7.6ab
SC
Curvilinear velocity (µm/s)
M AN U
Pre-freezing TTF-ULE/bear 77.6±5.1 46.8±6.8 Triladyl Canine® 72.0±6.8 41.5±4.5 CaniPro® 69.4±5.3 38.3±3.7 Extender 2 75.0±5.5 35.8±6.4 Post-thawing TTF-ULE/bear 71.8±8.2a 35.7±4.3a b 15.7±2.7b Triladyl Canine® 48.6±6.1 CaniPro® 46.3±8.8b 21.4±5.3b b Extender 2 47.9±8.3 20.6±4.5b a,b Rows with different superscripts differ (P < 0.05).
Average path velocity (µm/s)
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Progressive motility (%)
98.8±9.8a 53.0±8.3b 83.0±15.4ab 73.7±12.2ab
TE D
Total motility (%)
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Table 2. Sperm motility, kinetics, viability, acrosome integrity, and mitochondrial status (mean ± SEM) in brown bear semen according to the type of extender (n = 9).
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