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Theriogenology 66 (2006) 1797–1802

Influence of cooling rates and addition of Equex pasta on cooled

and frozen-thawed semen of generic gray (Canis lupus) and

Mexican gray wolves (C. l. baileyi)

C. Zindl a,b,*, C.S. Asa a, A.-R. Gunzel-Apel b

a Research Department, Saint Louis Zoo, 1 Government Drive, Saint Louis, MO 63110, USAb Institute for Reproductive Medicine, School of Veterinary Medicine, Bunteweg 15, 30559 Hannover, Germany

Abstract

A current priority for the preservation of the endangered Mexican gray wolf (Canis lupus baileyi) is the development of a sperm-

based genome resource bank for subsequent use in artificial insemination. To optimize the quality of cryopreserved sperm, the

procedures involved in processing semen before and during freezing need to be improved. The aim of this study were to examine the

effects of: (i) different cooling periods before freezing and (ii) addition of Equex pasta (Minitub, Tubingen, Germany) on the

characteristics of sperm from the generic gray wolf and the Mexican gray wolf after cooling and cryopreservation. For Mexican

wolf sperm, cooling for 0.5 and 1.0 h had a less detrimental effect on cell morphology than cooling for 2.5 h, whereas the slower

cooling rate (2.5 h) had a less detrimental effect on functional parameters and seemed to cause less damage to plasma membrane and

acrosome integrity than 0.5 and 1.0 h. For the generic gray wolf, cooling semen for 2.5 h had less detrimental effect on plasma

membrane integrity and viability; together with the 0.5 h cooling time, it yielded the highest percentages of intact acrosomes. As

previously shown in the domestic dog, Equex pasta had no beneficial effect on sperm characteristics in either wolf species.

# 2006 Elsevier Inc. All rights reserved.

Keywords: Generic gray wolf (Canis lupus); Mexican gray wolf (Canis lupus baileyi); Semen cryopreservation; Cooling period; Equex pasta

1. Introduction

Recovery of the Mexican gray wolf (Canis lupus

baileyi), a subspecies of the gray wolf (Canis lupus),

depends on careful genetic management of the captive

population. Because of their monogamous mating

system, transfer of gametes using cryopreservation

and artificial insemination is preferable to breaking

pair-bonds and transfer of animals. As there are only

few Mexican wolves available for evaluating the

* Corresponding author. Present address: Veterinary Clinic, Linden-

weg 8, 48734 Reken, Germany. Tel.: +49 17620083261;

fax: +49 2864900232.

E-mail address: claudia.zindl@gmx.net (C. Zindl).

0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2006.01.023

requisite technology, such as sperm cryopreservation,

it has been necessary to use the generic gray wolf as a

model and to base the techniques on those developed for

the domestic dog, the wolf’s closest relative. As cooling

is one of the critical steps in semen freezing, the effect

of various cooling rates on semen quality was compared

in both generic gray and Mexican wolves.

Sperm of several species require a pause of several

hours during cooling, before freezing, to develop

maximal resistance to the effects of freezing. In the

first studies regarding cooling and equilibration of

canine sperm [1], it was stated that none of the

temperature or time factors significantly affected the

revival rate. However, there have been a number of

studies investigating the effects of slow and rapid

C. Zindl et al. / Theriogenology 66 (2006) 1797–18021798

cooling of canine semen [2,3]. Some studies [4,5]

showed that the time taken during the cooling process is

an important factor. However, most studies have not

fully evaluated cooling rates or equilibration time

before freezing and have used arbitrary values [6].

Equex STM paste (Nova Chemical Sales, Scituate,

USA) was beneficial in dog semen cryopreservation; it

resulted in higher post-thaw survival, thermoresistance,

increased longevity and a higher proportion of sperm

with an intact plasma membrane [7,8]. In contrast,

Equex pasta (Minitub, Tubingen, Germany) did not

have the beneficial effect of Equex STM paste on

domestic dog semen [9]. The aim of the present study

was to investigate the effects of various cooling rates on

semen quality of both the generic gray and Mexican

wolf. Furthermore, the study was designed to explore if

sodium dodecyl sulphate (Equex pasta) had a beneficial

effect on wolf sperm.

2. Materials and methods

A total of 32 ejaculates (one ejaculate/week) from

four adult Mexican gray wolves (3 year of age) and 13

ejaculates (two ejaculates with a 2 day interval) of seven

generic gray wolves (3–6 year of age) was collected by

electroejaculation during the breeding season (January–

March) at the Wild Canid Research and Survival Center,

Eureka, MO, USA and the Wildlife Science Center,

Forest Lake, MN, USA, respectively.

The Mexican gray wolves were maintained outdoors

in pens of 312 m2 (three wolves) and of 215 m2 (two

wolves) near St. Louis, MO, in groups of two or three

animals, and fed a commercial dry chow diet (Mazuri

Exotic Canine Diet, PMI Nutrition International, St.

Louis, MO, USA; 1 kg/wolf/day). The generic gray

wolves were maintained in large outdoor pens near

Minneapolis, MN, USA, either in family packs or in

sibling groups of three to eight animals. They were fed

carcasses of white-tailed deer. In both locations, water

was provided ad libitum, and the wolves had valid

vaccination status.

For electroejaculation, the wolves were anesthetized

with ketamine (Ketaset1; Boehringer Ingelheim Vet-

medica Inc., St. Joseph, MO, USA; 4.2 mg/kg body

weight, i.m.) and xylazine (Rompun1; Bayer Corpora-

tion, Shawnee Mission, Kansas, USA; 2.3 mg/kg body

weight, i.m.) alone (generic gray wolves) or in

conjunction with isofluorane (1–3%; Isoflo1) (Mexican

gray wolves). Semen was collected by electroejacula-

tion using a Seager model ejaculator and a Platz no. 6

rectal probe, based on methods described by Platz and

Seager [10]. Semen of generic gray wolves were

collected at two collection days, semen of the Mexican

gray wolf was collected on seven collection days during

the 2001 breeding season. Following a successful

ejaculation series, one drop of semen was subjectively

examined for motility under a light microscope and

samples of similar quality from each animal were

pooled; color, volume, concentration and total sperm

number were recorded and the sample was centrifuged

at 1390 � g for 10 min at room temperature. Ejaculate

characteristics for generic and Mexican gray wolves

included volume (7.0 � 1.0 and 4.2 � 0.5 mL), con-

centration (271.7 � 59.4 �106 and 181.4 � 26.1 �106 sperm/mL) and total sperm count (1597.4 �390.4 � 106 and 756.2 � 153.9 � 106 sperm). After

dilution of the sperm pellet with a TRIS–egg yolk

(20%)-extender with 4% glycerol (modified from Ref.

[2]; 1 L contains 9.008 g Dextrose (Glucose), 24.228 g

TRIS (Tris[hydroxymethyl]aminomethane), 11.478 g

citric acid (anhydrous), 10,000 IU/mL penicillin and

10,000 mg/mL streptomycine (pH 7.45, 329 mOsM)) to

obtain a concentration of 40 � 106 sperm/mL (Mexican

and generic gray wolf) and 100 � 106 sperm/mL

(generic gray wolf, in case of a highly concentrated

semen sample), the extended semen was divided in three

aliquots into 15 mL tubes. Volumes of the samples for

generic and Mexican gray wolves were 5.1� 0.5 and

4.2� 0.4 mL, respectively and were cooled from 16 to

18 8C to 4 8C over 0.5, 1 or 2.5 h. The 0.5 h cooling time

was achieved by placing the extended samples directly in

the refrigerator at 4 8C, to achieve a cooling rate of 0.4–

0.5 8C/min (final temperatures for generic and Mexican

gray wolf semen samples were 5.5� 0.6 and

3.7� 0.5 8C, respectively). The 1 h cooling time was

achieved by placing the tube of extended semen in a

500 mL plastic beaker (9 cm in diameter), containing

250 mL of water as 16–18 8C, to achieve a cooling rate of

0.1–0.2 8C/min (final temperatures of generic and

Mexican gray wolf semen samples were 9.5� 0.7 and

7.5� 0.5 8C). The 2.5 h cooling time was achieved by

submersing the tube with extended semen in a 250 mL

glass beaker (diameter, 6.5 cm), containing 250 mL of

water at 16–18 8C, to achieve a cooling rate of 0.08–

0.1 8C/min (final temperature for generic and Mexican

gray wolf semen was 5.2� 0.4 and 3.2� 0.2 8C). After

completion of cooling of the three different semen

portions, the cooled portions were divided in two split-

samples and 1% Equex pasta (Minitub, Tubingen,

Germany) was added to one of the split-samples. Semen

was frozen in pellets (30 mL) on dry ice for 1 min, and

then transferred into liquid nitrogen for storage. Each

pellet was thawed in 1 mLTRIS-extender (no glycerol or

egg yolk) [2] at 37 8C until dissolution of the pellet.

C. Zindl et al. / Theriogenology 66 (2006) 1797–1802 1799

Table 1

Mean (�S.E.M.) percentages of sperm with an intact plasma membrane (HOST), normal morphology, intact acrosome, and of viable sperm in fresh,

cooled and post-thaw Mexican gray (C. l. baileyi) and generic gray wolf (C. lupus) semen

Intact plasma membrane Normal morphology Intact acrosome Viable sperm

Mexican gray wolf Fresh n = 27 89.1 � 1.3 73.9 � 4.7 71.9 � 4.7 96.0 � 0.9

Cooled n = 77 68.5 � 1.8C 69.5 � 2.5 57.5 � 2.8c 97.8 � 0.4

Post-thaw n = 143 57.3 � 1.3A,B 71.9 � 0.9 10.3 � 0.7A,B 79.0 � 1.3A,B

Generic gray wolf Fresh n = 13 86.7 � 1.3 77.0 � 3.4 71.9 � 6.8 98.8 � 0.4

Cooled n = 51 62.3 � 1.8C 72.9 � 2.4 62.5 � 2.9 99.8 �0.1

Post-thaw n = 98 40.7 � 2.0A,B 66.7 � 2.2 2.4 � 0.3A,B 51.5 � 2.3A,B

Superscripts in capital letters indicate highly significant differences (P < 0.01), whereas those in lower case indicate significant differences

(P < 0.05); (A/a, fresh-post-thaw; B/b, cooled-post-thaw and C/c, fresh-cooled).

Fig. 1. Mean (�S.E.M.) percentages of sperm with an intact plasma

membrane (HOST and 6-CFDA/PI), normal morphology, normal

spermatozoa with intact acrosome, viable sperm and forward motility

of fresh, cooled and frozen-thawed Mexican gray wolf (C. l. baileyi)

semen. Asterisk (*) indicates significant higher results (compared to

other cooling treatments) in the cooled and frozen-thawed group,

respectively (P < 0.05).

Semen quality was evaluated for fresh semen, after

cooling and again post-thaw. For evaluation of plasma

membrane integrity, a 100 mL aliquot from each sample

was incubated with 300 mL of hypo-osmotic solution

(60 mOsM fructose solution; Ref. [11]; 200 cells/slide)

in an Eppendorf tube at 37 8C for 45 min. Samples

processed in the field were fixed with 100 mL of an

18.5% formalin solution, pending microscopic evalua-

tion in the laboratory. For evaluation, a 10 mL drop of

the solution was placed on a slide and 200 sperm/slide

were evaluated under the microscope. A dual-fluor-

escent stain (propidium iodide and 6-carboxyfluores-

cein diacetate; Ref. [12] modified) was used to evaluate

the head plasma membrane integrity (200 cells/slide).

Slides stained in the field (fresh and cooled samples)

were mounted with Vectashield R Mounting Medium

(Vector Laboratories, Burlingame, CA, USA) to

preserve fluorescence during prolonged storage. For

morphology and acrosome assessment (200 cells/slide)

a 10 mL drop of semen was smeared on a slide and then

fixed and stained with Spermac1 (Sage Biopharma Inc.,

Bedminster, NJ, USA). Sperm were first classified as

morphologically normal or as abnormal head, mid-

piece or tail morphology; every normal and abnormal

cell was additionally classified having an intact, reacted,

swollen or lost acrosome. With a live-dead stain

(100 cells/slide) viability was assessed (Eosin-Nigro-

sin-stain; Society for Theriogenology, Hastings, NB,

USA). Furthermore, a 5 mL drop was subjectively

assessed for forward motility with a phase-contrast

microscope (four corners/slide for each sample).

2.1. Statistical analysis

The data were tested for normality using the Shapiro

Wilk W-test and were transformed with an ARCSIN

transformation. A full factorial ANOVA was used to

determine differences between treatments within a

species, with P < 0.05 and P < 0.01 regarded as

significant and highly significant, respectively.

3. Results

Mean (�S.E.M.) percentages of intact plasma

membrane, normal morphology, intact acrosome and

viable sperm in fresh, cooled and frozen-thawed

spermatozoa of Mexican gray and generic gray wolf

are presented in Table 1. Motility was assessed in fresh

and frozen-thawed samples, but not in cooled samples.

In both species, a highly significant decline (P < 0.01)

in the proportion of sperm with an intact plasma

membrane (HOST), of morphologically normal cells

with intact acrosomes, and of viable cells was found

after thawing in comparison to fresh and cooled samples

C. Zindl et al. / Theriogenology 66 (2006) 1797–18021800

Fig. 2. Mean (�S.E.M.) percentages of sperm with intact plasma

membrane (HOST and 6-CFDA/PI), normal morphology, normal

spermatozoa with intact acrosomes and viable spermatozoa of fro-

zen-thawed Mexican (C. l. baileyi) and generic gray wolf (C. lupus)

semen, supplemented with or without 1% Equex pasta.

(Fig. 1). The percentage of sperm with intact plasma

membranes in cooled semen of both species was

substantially lower (P < 0.01) and percentage of

spermatozoa with intact acrosomes of cooled Mexican

wolf semen was lower (P < 0.05) in comparison to

fresh semen (Table 1). Forward motility decreased

significantly during cryopreservation (Fig. 1).

Mexican wolf semen cooled for 1 h had a higher

percentage of sperm with normal morphology and intact

acrosome in comparison to samples cooled for 2.5 h

(P < 0.05; Fig. 1). In both wolf species, semen

containing 1% Equex pasta was of lower quality

(Fig. 2 and Table 2). Samples cooled for 2.5 h, and in

particular samples without 1% Equex pasta and cooled

for 2.5 h, had higher percentage of sperm plasma

Table 2

Mean (�S.E.M.) percentages of sperm with intact plasma membrane (HOST

spermatozoa in post-thaw Mexican gray (C. l. baileyi) and generic gray w

Intact plasma membrane

HOST 6-CFDA/

Mexican gray wolf With E n = 67 51.2 � 1.9** 47.8 � 2.

Without E n = 76 62.2 � 1.5** 58.9 � 1.

Generic gray wolf With E n = 47 32.4 � 2.8** 22.6 � 2.

Without E n = 51 48.2 � 2.4** 32.7 � 3.

* Differences (P < 0.05).** Differences (P < 0.01).

membrane integrity (6-CFDA/PI) in post-thaw evalua-

tion than samples cooled for 0.5 h or 1 h (P < 0.05;

Figs. 1 and 2).

4. Discussion

This is the first study to investigate effects of

different cooling rates and addition of sodium dodecyl

sulphate on Mexican and generic gray wolf semen. In

both species, the quality of semen deteriorated after

cooling. After cooling, semen of both species cooled for

1 h only reached a final temperature of <7–9 8C; this

may account for the better quality in comparison to the

samples reaching refrigerator temperature. Further-

more, they may have entered the freezing period in

better condition (with respect to plasma membrane

integrity and intact acrosomes), than samples cooled for

0.5 or 2.5 h. An explanation may be the phase transition

temperature, the temperature when membrane lipid

phase transitions in sperm occur [13–16]. At this

temperature, the membrane’s lipid phase changes from

a liquid-crystalline to a gel state, or vice versa, having

profound effects upon membrane properties. In boars

[15], when the temperature was reduced below the

phase transition temperature, the resulting lateral phase

separations were considered as a mechanism of cold-

induced cell injury. The phase transition temperatures

for boar sperm, for example, are at 32 and 6 8C [17], but

these have not yet been established for canine semen.

Post-thaw, samples cooled in 2.5 h had better results.

Conversely, samples cooled over 1 h were frozen from a

temperature higher than the conventional 4–5 8C, and

thus were subjected to a faster freezing rate below 7–

9 8C; this may have resulted in the poor post-thaw

quality of these samples.

Considering only the frozen-thawed samples without

addition of 1% Equex pasta in this study, cooling for

2.5 h had the most beneficial effect on functional and

and 6-CFDA/PI), normal morphology, intact acrosome and of viable

olf (C. lupus) semen, treated with or without 1% Equex pasta (E)

Normal morphology Intact acrosome Viable sperm

PI

1** 69.6 � 1.4* 8.9 � 1.0 78.1 � 2.2

8** 73.8 � 1.3* 11.4 � 1.0 79.7 � 1.5

7* 68.6 � 3.1 1.5 � 0.3** 48.3 � 3.4

1* 65.8 � 3.1 3.1 � 0.5** 54.5 � 3.2

C. Zindl et al. / Theriogenology 66 (2006) 1797–1802 1801

viability parameters of Mexican and generic gray wolf

semen. Because the samples cooled for 2.5 h had the

best results for intact head and tail plasma membranes

and highest viability after the freeze-thaw process, this

slower cooling may not induce cold-shock as does a

more rapid cooling rate. In contrast, Martin [1] reported

that none of the temperature or time factors significantly

affected the revival rate. Bateman [18] also found that

post-thaw results were not affected by different cooling

rates. Pukazhenthi [19] found significant acrosomal

damage in cat semen; the extent of temperature induced

damage was significantly mitigated by slow cooling

(0.5 8C/min). The results of the present study were

consistent with the view of Watson [20] who suggested

that the longer cooling period allowed time for

membrane changes or ionic fluxes to occur, which

rendered membranes more resistant to cooling. Oettle

[5] observed that a significant amount of damage to the

acrosome appears to occur during cooling of dog semen.

This view was supported by Hay et al. [3] who

compared dog semen samples that were cooled quickly

(0.5 h) or slowly (3 h) to 0 8C; they found that slow

cooling did not adversely affect ability to penetrate

oocytes. However, Goodrowe et al. [21] observed no

differences between cooling treatments of red wolf

(Canis rufus) semen either after cooling or freezing

and thawing.

Samples supplemented with 1% Equex pasta did not

have as good results after freeze-thawing as the samples

without Equex pasta. The effects of sodium dodecyl

sulphate on canine semen characteristics were inves-

tigated by some authors [7,8]. The reason for not

detecting the same beneficial effect described by Refs.

[7,8], who used Equex STM paste (Nova Chemical

Sales, Scituate Inc., MA, USA) as a source of sodium

dodecyl sulphate might be that the effect of Equex STM

paste and Equex pasta is different [9]. Samples

supplemented with Equex STM paste had significantly

higher post-thaw motility and live cells with intact

acrosomes, in comparison to samples supplemented

with Equex pasta or unsupplemented controls. The

efficiency of Equex STM paste may be related to the

protective effect on the sperm membranes (against lipid

phase transitions, etc.) and might also partially protect

the functionality of the plasma membrane Ca2+-pumps,

but caused a higher intracellular Ca2+ concentration

instead of lowering it. The result that a beneficial effect

of Equex pasta was negligible may have been because it

was not added to the extender in the same concentration,

and not at the same stage during the preservation

process. The final concentration of 0.5% was found not

to be beneficial [9] and the final concentration of 1% as

tested in this study did not have the expected positive

effect. Another explanation may be that wolf sperm

plasma membranes react differently to sodium dodecyl

sulphate than the membrane of dog sperm. Additionally

Equex paste was added directly in the diluted semen,

instead of being mixed with the extender; this can lead

to immediate damage of the plasma membrane because

of the osmotic capability of being a detergent.

For cryopreservation of Mexican and generic gray

wolf semen, data from the present study supported a

slow cooling rate (0.08–0.1 8C/min) to reach refrig-

erator temperature (i.e. 5 8C) before freezing. The

addition of 1% Equex pasta is not recommended. It

would be of interest to investigate the effect of 0.5%

Equex STM paste or a different final concentration of

Equex pasta and of different equilibration periods of the

cooled semen samples to improve the wolf semen

cryopreservation protocol.

Acknowledgements

This work was supported by the Deutscher Akade-

mischer Austauschdienst (DAAD), Germany. The

authors thank the St. Louis Zoo Repro Lab team and

the teams at the Wild Canid Research and Survival

Center, Eureka, MN, USA and at the Wildlife Science

Center, Forest Lake, MN, USA.

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