CHARACTERIZATION AND CRYOPRESERVATION

OF SEMEN FROM ENDANGERED MARKHOR GOATS

(CAPRA FALCONERI HEPTNERI) WITH EVALUATION OF
REPRODUCTIVE SEASONALITY
Author(s): Marisa Bezjian , D.V.M., Noha Abou-Madi , D.V.M., Dipl. A.C.Z.M.,
George V. Kollias , D.V.M., Ph.D., Dipl. A.C.Z.M., John E. Parks , Ph.D., Soon
Hon Cheong , D.V.M., Ph.D., Dipl. A.C.T. and Katherine A. Beltaire , D.V.M.,
Dipl. A.C.T.
Source: Journal of Zoo and Wildlife Medicine, 44(3):672-685. 2013.
Published By: American Association of Zoo Veterinarians
DOI: http://dx.doi.org/10.1638/2012-0267R.1
URL: http://www.bioone.org/doi/full/10.1638/2012-0267R.1

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 Journal of Zoo and Wildlife Medicine 44(3): 672–685, 2013
Copyright 2013 by American Association of Zoo Veterinarians

CHARACTERIZATION AND CRYOPRESERVATION OF SEMEN

FROM ENDANGERED MARKHOR GOATS (CAPRA FALCONERI
HEPTNERI) WITH EVALUATION OF REPRODUCTIVE
SEASONALITY

Marisa Bezjian, D.V.M., Noha Abou-Madi, D.V.M., Dipl. A.C.Z.M., George V. Kollias, D.V.M., Ph.D.,
Dipl. A.C.Z.M., John E. Parks, Ph.D., Soon Hon Cheong, D.V.M., Ph.D., Dipl. A.C.T., and Katherine
A. Beltaire, D.V.M., Dipl. A.C.T.

Abstract: The aims of this study were to determine the reproductive seasonality of four captive markhor goats
(Capra falconeri heptneri), to characterize semen collected by electroejaculation, and to compare extenders and
processing techniques for semen cryopreservation. Over the course of 1 yr, mean monthly scrotal circumference,
serum testosterone, and fecal testosterone were measured and found to be inversely associated with day length.
Maximum scrotal circumference (25.2 6 0.9 cm), serum testosterone (521.0 6 103.4 ng/dl), and fecal testosterone
(382.5 6 90.3 ng/g) occurred in November, when day length was short (9.7 6 0.1 hr). Once a month for 3 mo
(December, January, and February), bucks were anesthetized for electroejaculation and semen evaluation. Semen
samples were divided into six aliquots for extension and cryopreservation in soy-based Bioxcellt or Tris-based
extender with 5 or 15% egg yolk, with and without centrifugation. Samples were then thawed for repeat evaluation
1–3 mo later. Postthaw evaluation revealed no significant differences between centrifuged and noncentrifuged
samples. Sperm in Tris 5% and 15% egg yolk displayed higher total motility at 0, 3, and 6 hr postthaw and higher
progressive motility postthaw compared with sperm in Bioxcell (P , 0.05). Sperm in Bioxcell displayed higher
viability than sperm in both Tris–egg yolk extenders (P , 0.01), more intact acrosomes than sperm in Tris–15% egg
yolk (P , 0.05), and a tendency for more intact acrosomes than sperm in Tris–5% egg yolk (P , 0.10). Sperm in
Tris–5% egg yolk tended to have a higher percentage of morphologically normal sperm compared with Bioxcell (P
, 0.10). This study provides evidence that markhor goats exhibit seasonality in scrotal circumference and
testosterone levels and that centrifugation may be eliminated from the processing of markhor semen.
Key words: Capra falconeri heptneri, markhor, sperm cryopreservation, reproductive seasonality.

INTRODUCTION ongoing.62 In situ conservation efforts have fo-

Markhor goats (Capra falconeri) inhabit semiar-
id, mountainous terrain in Afghanistan, Pakistan,
Tajikistan, India, Turkmenistan, and Uzbekistan
(22–418N, 59–798E).20,33,51,60 Since 1994, markhor
goats have been classified as endangered by the
International Union for Conservation of Nature
and Natural Resources due to poaching for meat
and trophy horns, competition for land with
domesticated livestock and logging ventures, and
civil unrest.33,60 The number of markhor goats may
actually be greater than the current estimate of
,2,500 mature individuals worldwide.60 Recent
surveys have indicated increases in markhor
numbers by as much as 50% in Pakistan and
Tajikistan where conservation efforts have been
From the Department of Clinical Sciences, College of
Veterinary Medicine (Bezjian, Abou-Madi, Kollias,
Cheong, Beltaire) and the Department of Animal Science,
College of Agriculture and Life Sciences (Parks), Cornell
University, Ithaca, New York 14853, USA. Correspon-
dence should be directed to Dr. Beltaire (kbeltaire@
vasci.umass.edu).
cused on preserving markhor habitats, limiting
hunting and illegal trade, and providing incentives
for community-based land and wildlife steward-
ship, whereas ex situ conservation efforts have
focused on captive breeding programs.20,33,60 For
other endangered species, ex situ efforts have
been supported by assisted reproductive tech-
niques (ARTs), such as artificial insemination
(AI) and semen cryopreservation.1,27,54–56
AI with cryopreserved semen limits the cost,
stress, health problems, and disease transmission
risks associated with transportation of live ani-
mals for breeding.1,39 In addition, cryopreserva-
tion and long-term storage of semen preserve the
genetics of valuable, underrepresented, geograph-
ically isolated, and even deceased males.1,27,39 This
is particularly important for markhor goats that
exist in fragmented, small populations in the wild
(,100 individuals) or in extremely small groups in
zoos.20,60 ARTs can be useful for improving the
genetic integrity of populations, but implementa-
tion should be preceded by genetic analysis of the
candidate goats. From 1996 to 1998, a study at
three European zoos revealed that 35.7% of

672
 BEZJIAN ET AL.—MARKHOR GOAT SEMEN AND REPRODUCTIVE SEASONALITY
markhor were introgressed by domestic goat
mitochondrial DNA.20 In 1999, the American
Zoo Association’s Caprinae Taxon Advisory
Group issued a report regarding analysis of
mitochondrial DNA from 16 markhor in four
North American zoos.48 In that report, 75% of the
markhor were grouped into a single clade identi-
fied as Capra falconeri heptneri. The remaining
25% were grouped into the wild goat clade Capra
aegagrus, from which the domestic goat (Capra
hircus) is thought to be derived. The report
concluded that the presence of presumptive C.
aegagrus lineage may be the result of hybridization
in the distant past and part of the normal
variability of wild markhor.48
To apply ARTs most effectively, the basic
reproductive physiology of markhor goats must
be known. Many caprine species from high
latitudes (.358N) and some subtropical latitudes
(25–358N) display increased libido, testicular size,
testosterone production, and sperm production
during the breeding season.10,12–14,29,36,44–47,65 These
reproductive patterns are typically inversely re-
lated to photoperiod, making the goats short day
length breeders.12–14,29,36,44–47,65 Because testosterone
is required for both sperm production and libido,
studies have evaluated changes in serum testos-
terone or fecal testosterone to track reproductive
seasonality.17,23,28,35,47,58 Scrotal circumference also
has been used to investigate reproductive patterns
because it increases during the reproductive
season for many species and is closely correlated
with testosterone, sperm production, number of
females impregnated, and early puberty and
ovulation rate in female offspring in domestic
small ruminants.11,17,34,35,41,65
Cryopreservation of sperm for long-term stor-
age and use in AI programs is a complex process
involving consideration of extender, processing
method, cooling rate, freezing method, and spe-
cies-specific semen composition.21 For domesti-
cated caprine species, the most commonly used
extenders contain glycerol and either egg yolk or
nonfat, dried skim milk.29,30 A challenge specific to
the freezing of caprine semen is the presence of
lipases produced in the buck bulbourethral gland
that hydrolyze egg yolk lecithin and milk triglyc-
erides into sperm-toxic fatty acids and lysoleci-
thin.24,29,43,47 Many studies have been performed on
techniques to coat, wash, or centrifuge semen to
limit the contact of these bulbourethral gland
enzymes with sperm.10,23,54,55,63,64,66 There is also a
need to find extenders that do not contain animal
proteins due to concerns regarding bacterial or
viral transmission and variability between batches
673
of extenders containing egg yolk or milk.61
Extenders such as Bioxcellt that use soybean
(Glycine max) (or plant-derived) protein have been
demonstrated to be suitable cryoprotectants for
freezing semen from ruminants.16,27,57,61
To date, there have been no published studies
on the reproductive seasonality of markhor bucks
or the methods involved in markhor semen
collection, extension, and cryopreservation.
Therefore, the present study was performed to 1)
evaluate monthly changes in day length and
captive markhor buck scrotal circumference,
serum testosterone, and fecal testosterone over
the course of 1 yr; 2) characterize ejaculates (e.g.,
volume, color, pH, sperm viability, motility,
concentration, morphology, and acrosome integ-
rity) from markhor bucks collected via electro-
ejaculation once a month for 3 mo; and 3)
compare extenders (Bioxcell or Tris-based ex-
tender with 5 or 15% egg yolk) and processing
techniques (with and without centrifugation) for
markhor semen cryopreservation.
MATERIALS AND METHODS
Goats and husbandry
The Cornell University Institutional Animal
Care and Use Committee approved all proce-
dures, including the use of animals in this study.
Four male markhor goats (C. f. heptneri), ranging
in age from 8 to 10 yr old were enrolled in this
study. The bucks were all born in captivity at the
Rosamond Gifford Zoo in Syracuse, New York,
USA, and had never been used for breeding. All
four males shared the same sire, whereas bucks 1–
3 shared the same dam. Bucks 2 and 3 were twins.
The bucks were housed together in a sloped
terrain exhibit (1,860 m2) composed of lime-
stone-quarried products, rocks, gravel, and stone
dust from limestone cuttings. Bucks were exposed
to natural annual light cycles, with no supplemen-
tation with artificial light. Four indoor barn-style
holding areas and a chute system were attached to
the management area at the back of the exhibit.
Food was offered in the holding area, but bucks
had free access to the exhibit. An adult female
markhor goat, the dam to bucks 1–3, was housed
in an adjacent exhibit that allowed visual, audito-
ry, and olfactory contact with the bucks but no
physical contact. The bucks had established their
own hierarchy, so sparring was seen infrequently.
Each buck was offered free choice water and
approximately 0.9 kg of a high-fiber pellet sup-
plement (Mazurit ADF-#25 Herbivore, Land
O’Lakes, Inc., Saint Paul, Minnesota 55164,
674 JOURNAL OF ZOO AND WILDLIFE MEDICINE
USA) and 250 ml of rice bran pellet (Equi-Jewelt,
Kentucky Performance Products, LLC, Versailles,
Kentucky 40383, USA) once a day. Grass hay (1.4
kg, second cutting) was provided for each buck
twice daily.
Routine health management consisted of annu-
al physical examination with blood collection,
tuberculin skin test using PPD Bovis (Colorado
Serum Co., Denver, Colorado 80216, USA; 0.1 ml
i.d.), fecal culture surveillance for Johne’s disease,
and vaccination against Clostridium perfringens
type C & D with tetanus toxoid (Colorado Serum;
2 ml s.c.) and rabies (Imrabt 3, MERIALt,
Duluth, Georgia 30096, USA; 2 ml s.c.). Markhor
goats were supplemented annually with BO-SEt
(Schering-Plough Animal Health, Union, New
Jersey 07083, USA; 0.06 mg/kg s.c.) and vitamin
E 300 (AgriLabs, St. Joseph, Missouri 64505,
USA; 40 IU/kg s.c.), and dewormed with Iver-
mectin-1% (IVOMECt, MERIAL; 0.2 mg/kg
s.c.). Quarterly fecal analyses were performed to
assess the efficacy of the deworming protocol.
Each buck was identified with a colored and
numbered ear tag.
Day length
To track the annual cycle of day length from
October 2010 through September 2011 at the
Rosamond Gifford Zoo in Syracuse (438N, 768W),
daily values of sunrise and sunset were collected
from the United States Naval Observatory Astro-
nomical Applications Department.59 These data
were then used to calculate individual day length
and mean monthly day length.
Blood and fecal collection and scrotal
circumference measurement
Once a month from October 2010 through
September 2011, the bucks were herded into the
chute system attached to the back of their main
exhibit. For nine of the 12 mo, the bucks were
manually restrained for lateral saphenous or
cephalic venipuncture and scrotal circumference
measurement by using a metal scrotal measuring
tape. For the remaining 3 mo (December–Febru-
ary), sampling was performed under chemical
restraint (as described below). All scrotal circum-
ference measurements were taken by the primary
author (MB) without evaluation of previous
measurement data to achieve blinding. Fresh fecal
samples were obtained monthly by direct obser-
vation with animal identification. Samples were
collected on the 15th day of the month (65 days).
Whole blood samples were allowed to clot for 30
min at room temperature and then centrifuged
(VanGuard #6500; Hamilton Bell Co., Inc., Mon-
tvale, New Jersey 07645, USA; maximum centrif-
ugal force ¼ 1,318 g) for 10–15 min to separate the
serum. Serum and fecal samples were stored at
708C for the duration of the study. At the end of
the 12 mo, frozen samples were shipped to the
Smithsonian Conservation Biology Institute Cen-
ter for Species Survival (National Zoological
Park, Front Royal, Virginia 22630, USA) for
serum and fecal steroid extraction and quantita-
tive measurement of testosterone using the Coat-
A-Countt (CAC) Total Testosterone, a solid-
phase radioimmunoassay (Siemens, San Francis-
co, California 94080, USA). Intraassay and inter-
assay coefficients for the CAC used were ,10 and
15%, respectively. Frozen fecal samples were
placed into a lyophilizer for 5 days to remove
the water content. Parallelisms were run to
validate the Total Testosterone CAC assay to
determine the appropriate dilution of 1:10 for the
total testosterone assay.
Semen collection
Before anesthesia for semen collection, food
and water were withheld for 12–24 hr. The
markhor bucks were individually run through
the chute system, and each buck was given an
injection of medetomidine hydrochloride (Zoo-
Pharm, Fort Collins, Colorado 80522, USA; 0.05
mg/kg i.m.) and ketamine hydrochloride (KETA-
VED , Vedco, Saint Joseph, Missouri 64507,
TM
USA; 2 mg/kg i.m.) for anesthesia. After induc-
tion, each buck was transported into an enclosed
barn and positioned in right lateral recumbency.
Vital signs that were monitored included heart
rate, respiratory rate, rectal temperature, and
noninvasive blood pressure, and supplemental
nasal oxygen was provided. Each buck received
1–2 L of subcutaneous Plasmalyte-A (Baxter,
Deerfield, Illinois 60015, USA) to promote anes-
thetic agent elimination and to help maintain
hydration. Blood was collected from the left
lateral saphenous vein, and scrotal circumference
was measured with the same metal scrotal mea-
suring tape. Palpation and ultrasound evaluation
(SonoSite Titan [C60/5-2 MHz broadband curved
array], Bothell, Washington 98021, USA) of the
scrotum, testes, epididymides, and spermatic
cords did not reveal any significant anatomic
abnormalities that would affect sperm produc-
tion. Before semen collection, the penis was
extended from the prepuce to minimize bacterial
contamination that could alter the freeze-thawing
process.37,56 Manual extension of the penis was
 BEZJIAN ET AL.—MARKHOR GOAT SEMEN AND REPRODUCTIVE SEASONALITY
initially attempted with bucks in lateral recum-
bency. Because this approach was unsuccessful,
bucks were elevated vertically into a ‘‘sitting’’
position to apply additional pressure to the
sigmoid flexure. Once the urethral process was
visualized and manipulated, the penis was extrud-
ed from the prepuce and held in extension with
gauze. This vertical position was not maintained
for .6 min per attempt. Bucks were slowly
lowered back into right lateral recumbency for
semen collection.
Electroejaculation was performed with a probe
(Bailey Ejaculator Mod 2, Western Instrument
Co., Denver, Colorado 80216, USA) coated with
nonspermicidal lubricant (Priority Caret, First
Priorityt, Inc., Elgin, Illinois 60123, USA). Bucks
were massaged rectally with the probe for approx-
imately 10–15 sec before electrical stimulation.
This probe had two bipolar ring electrodes
approximately 2.4 cm in diameter with a 1.0-cm
spacer between them, and an output of approxi-
mately 5 V peak to peak. Stimulations lasted 4–6
sec, with pauses of 4–6 sec between stimulations.
Bucks varied with respect to the number of ‘‘on-
and-off’’ cycles needed to induce ejaculation from
approximately three to six cycles. Semen was
collected into a clean, warmed plastic bag within
an insulating glove. After semen collection, keto-
profen (Ketofent, Fort Dodge Animal Health,
Fort Dodge, Iowa 50501, USA; 2 mg/kg s.c.) was
administered for anti-inflammatory and analgesic
effects, and anesthesia was reversed with atipa-
mezole (Antisedant, Pfizer Animal Health, Mad-
ison, New Jersey 07940, USA; 0.15 mg/kg i.m.).
Semen characterization
Immediately after collection, the color and
volume of each ejaculate were evaluated. Percent-
ages of total sperm motility (TM) and progressive
sperm motility (PM) were determined by diluting
5 ll of raw semen with 10–20 ll of prewarmed
(378C) 2.9% sodium citrate on a prewarmed slide
at 3200 magnification over multiple fields.65
Sperm concentration was measured using a he-
mocytometer (Hausser Scientific, Horsham,
Pennsylvania 19044, USA), and the pH of each
ejaculate was determined using pH paper (pHy-
driont, Micro Essential Laboratory, Inc., Brook-
lyn, New York 11210, USA).2 For assessment of
sperm morphology and viability, 5 ll of raw
semen was mixed with 500 ll of 2.9% sodium
citrate, and 1 drop of this dilution was added to a
glass slide with 1 drop of eosin–nigrosin stain
(Society of Theriogenology, Hastings, Nebraska
68901, USA), smeared, and 100 cells were evalu-
675
ated at 31,000 magnification under oil immer-
sion.4 For assessment of acrosome integrity, a
smear of the 1:100 dilution of each ejaculate was
prepared, air-dried, and stained with Spermact
stain (Minitube of America, Verona, Wisconsin
53593, USA), and then 100 cells were assessed for
intact or altered (reactive and defective) acro-
somes at 31,000 magnification under oil immer-
sion.8,25,42
Semen cryopreservation
Good-quality semen samples were needed to
determine the effects of different extenders and
processing methods on markhor sperm response
to cryopreservation; so, the inclusion criteria for
raw semen samples were as follows: 1) TM  50%,
2) PM  40%, and 3) no gross contamination or
agglutination. Based on prior studies with other
ruminant species, the following three extenders
were selected for cryopreservation of markhor
sperm: 1) Bioxcell (soybean lecithin extender,
IMV Technologies, Maple Grove, Minnesota
55369, USA); 2) Tris–5% egg yolk, containing
250 M Tris-(hydroxymethyl)aminomethane, 81 M
citric acid, 69 M glucose, and 5% egg yolk (v/v);
and (3) Tris–15% egg yolk containing 250 M Tris-
(hydroxymethyl)aminomethane, 81 M citric acid,
69 M glucose, and 15% egg yolk (v/v). Chemicals
for the Tris-based extenders were purchased from
Sigma-Aldrich (Saint Louis, Missouri 63178,
USA), and fresh specific-pathogen-free (SPF)
eggs were supplied by the Poultry Facility man-
aged by the Department of Microbiology and
Immunology at the Cornell University College of
Veterinary Medicine, Ithaca, New York, USA.
Each ejaculate was divided into six equal
aliquots. Three of the aliquots were diluted with
the extenders described above to a concentration
of 200–400 3106 sperm/ml and cooled to 48C. The
remaining three aliquots were diluted 1:10 (v/v)
with the extenders described above and centri-
fuged immediately postdilution to minimize sem-
inal lipase interaction with egg yolk at 1,318 g for
7 min. The seminal plasma supernatants were
discarded, and the sperm pellets were resus-
pended with the appropriate extender to a con-
centration of 200–400 3 106 sperm/ml and then
cooled to 48C. The aliquots were placed in a bath
of approximately 50 ml of distilled water at 378C
and into a refrigerator for controlled cooling of
samples to 48C .2 hr. The samples were then
stored at 48C until transportation from the zoo to
the laboratory for freezing. The time from initia-
tion of cooling to freezing was standardized at
approximately 5 hr. After cooling, extenders
676 JOURNAL OF ZOO AND WILDLIFE MEDICINE
described above with 14% glycerol (v/v) cooled to
48C were added in four fractions at 15-min
intervals over 1 hr to achieve a final concentration
of 100–200 3 106 sperm/ml in 7% glycerol.
Extended semen was then equilibrated for 1 hr
at 48C. The importance and length of glycerol
equilibration varies by species and individual
male. Because of potential osmotic effects and
low glycerol permeability, an equilibration period
of 1 hr was deemed worthwhile. Percentages of
TM and PM were again assessed before freezing
by evaluation of 10 ll of the equilibrated semen
on a prewarmed slide at 3200 magnification over
multiple fields. Semen was frozen in 0.25-ml
straws at 4 cm above the surface of liquid nitrogen
for 10 min. After freezing, the straws were
plunged and then stored in liquid nitrogen for 1–
3 mo.
Postthaw evaluation
Thawing was performed in a 378C waterbath for
30 sec. Percentages of TM and PM were evaluated
at 0, 3, 6, 9, and 12 hr postthaw, with storage of
semen in a 378C incubator between evaluations.
Morphology, viability, and acrosome integrity
were evaluated at 0 and 3 hr postthaw. To evaluate
morphology and viability, 50 ll of the thawed
semen was added to a glass slide with 1 drop of
eosin–nigrosin stain and assessed as described
previously.4 To evaluate acrosome status, 50 ll of
thawed semen was spread on a glass slide, air-
dried, stained with Spermac stain (Minitube of
America), and assessed as described previously.
All postthaw samples were evaluated by the
trained primary author (MB) in a random se-
quence to achieve blinding. Evaluations were
supervised by the last author (KB).
Statistical analysis
Statistical analysis of seasonality was per-
formed using IBM SPSS Statistics 20 (Interna-
tional Business Machines Corp., Armonk, New
York 10504, USA). Bivariate associations among
measurements taken monthly for 1 yr were
assessed via linear mixed models, taking into
account the nonindependence of observations
via a random effect for each individual buck.
Model assumptions were checked in each instance
(including normality of residuals and homogene-
ity of variances) and were found to be acceptable.
Using this statistical methodology, it was possible
to control for the correlation of measurements
within each buck over time and to estimate the
associations between pairs of continuous vari-
ables across all bucks, analogous to bivariate
correlation or regression.
Statistical analysis of extender and processing
method was performed using JMP Pro-version 9
(SAS Institute Inc., Cary, North Carolina 27513,
USA). A two-way mixed model analysis of
variance was used to evaluate differences between
extenders (Bioxcell, Tris–5% egg yolk, and Tris–
15% egg yolk) and processing methods (with and
without centrifugation) on sperm motility at
different time points (postequilibration and post-
thaw 0, 3, 6, 9, and 12 hr) and sperm viability,
acrosome integrity, and morphology at 0 and 3 hr
postthaw. To account for variability between
individual samples, the model included time
nested within collection date nested within indi-
vidual buck. Interactions were tested and includ-
ed in the final model that was built using a
backwards stepwise method. The effects of indi-
vidual extender and processing method were
evaluated using post hoc contrast of least-squares
means with Bonferroni correction for multiple
testing. The sample size used for statistical
evaluation included a total of five semen samples.
The remaining six samples did not meet the
inclusion criteria or had preliminary differences
in laboratory methods. Total motility, progressive
motility, viability, acrosome integrity, and normal
morphology were arcsine transformed to fulfill
the normal distribution assumption. Values were
reported as mean 6 SEM, and P , 0.05 was
considered significant. Due to the limitation of a
small sample size, this study also presents param-
eters with P , 0.10 for consideration as tenden-
cies or trends.
RESULTS
Reproductive seasonality
From October 2010 to September 2011, mean
monthly day length in Syracuse followed a
Northern hemisphere seasonal pattern of decreas-
ing through the autumn to a minimum in the
winter (9.1 6 0.0 hr in December), and then
increasing through the late winter and spring to a
maximum in the summer (15.3 6 0.0 hr in June)
(Fig. 1). Significant inverse associations were
found between mean monthly day length and
scrotal circumference (b ¼ 0.474, SE ¼ 0.113, P
, 0.001), mean monthly day length and serum
testosterone (b ¼ 0.005, SE ¼ 0.002, P , 0.05),
and mean monthly day length and fecal testoster-
one (b ¼ 0.006, SE ¼ 0.003, P , 0.05). Mean
scrotal circumference and testosterone increased
rapidly through the autumn when day length was
 BEZJIAN ET AL.—MARKHOR GOAT SEMEN AND REPRODUCTIVE SEASONALITY 677

Figure 1. Monthly mean (6SEM) day length (hours) () and markhor goat (n ¼ 4) scrotal circumference (cm) (*)
from October 2010 through September 2011.

decreasing. Maximum scrotal circumference (25.2
6 0.9 cm), serum testosterone (521.0 6 103.4 ng/
dl), and fecal testosterone (382.5 6 90.3 ng/g)
occurred in November (9.7 6 0.1 hr day length).
Scrotal circumference then decreased through the
winter and spring to a nadir of 18.4 6 0.5 cm in
May (Fig. 1). After peaking in November, both
serum and fecal testosterone decreased through
the winter and then exhibited minor fluctuations
(Fig. 2). Changes in serum and fecal testosterone
were directly correlated (b ¼ 0.536, SE ¼ 0.060, P
, 0.001), thus serum and fecal testosterone
followed a similar annual cycle.
Semen characterization
A total of 11 ejaculates were collected from the
four markhor bucks during December, January,
and February (Table 1). Samples varied between
months and individual bucks. In February, buck 4
was not used for collection due to inability to
extrude the penis from within the prepuce.
Sample collection was abandoned as ejaculation
within the prepuce could cause sample contami-

Figure 2. Monthly mean (6SEM) serum testosterone (ng/dl) () and fecal testosterone (ng/g) (*) for markhor
goats (n ¼ 4) from October 2010 through September 2011.
678 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Table 1. Characterization of raw semen samples (n ¼ 11) collected by electroejaculation of four captive markhor bucks in December 2010, January 2011, and

White
Table 2. Characterization of raw semen samples (n

1.20
1.16
1.39
Jan

55
45
32
74
55
7.0
¼ 9)a collected by electroejaculation of four captive
Buck 4 markhor bucks in December 2010, January 2011, and
February 2011.

White
1.40
4.29
6.01
Dec

80
65
45
58
58
7.0
Variable Mean 6 SEM

Volume (ml) 1.2 6 0.2

Concentration (3 109/ml) 2.4 6 0.3
White
0.50
2.55
Feb

Total sperm (3 109) 3.0 6 0.7

40
30
63
73
68
7.2
1.3

pH 7.4 6 0.2
Total motility (%) 60.6 6 6.4
Progressive motility (%) 50.0 6 5.5
Buck 3

White
1.75
2.93
5.13
Jan

85
75
61
74
66
Viable (live) sperm (%) 48.9 6.0
7.1

6
Normal morphology (%) 65.9 6 4.6
Intact acrosome (%) 66.6 6 3.7
White
1.00
3.17
3.17
Dec

a
Samples with urine contamination or gel agglutination were
80
60
56
56
79
8.2

excluded from this table.

nation and dilution. Also in February, urine

Yellow
10.25
0.03
0.28
Feb

8.2

69
15
57
5
0

contamination was observed during sample col-

lection from buck 2, and gel agglutination was
detected in the semen sample collected from buck
White

1. Exclusion of those samples yielded the results

0.48
2.24
1.07
Jan

50
40
36
73
67
7.2
Buck 2

displayed in Tables 2 and 3. In the remaining nine

raw semen samples, there were 65.9 6 4.6%
morphologically normal sperm, and the most
White-yellow

common defects were bowed midpieces (11.6 6

1.20
1.50
1.80
Dec

55
50
60
38
83

3.9%), bent tails (9.3 6 1.1%), and detached heads

7.0

(5.3 6 1.7%) (Fig. 3).

Semen cryopreservation
Yellow
0.45
1.83
0.82

Raw semen samples from bucks 1–4 in Decem-

Feb

30
15
48
37
50
8.0

ber and bucks 2–4 in January met the inclusion

criteria for evaluation of extender and processing
method. However, samples from bucks 2 and 3 in
White-yellow
2.50
2.31
5.78
Jan

30
25
16
62
49
7.8

Table 3. Abnormal sperm morphology in raw

Buck 1

semen samples (n ¼ 9)a collected by electroejaculation

of four markhor bucks.

Morpholgic defect Mean 6 SEM (%) Range (%)

White-yellow

Detached head 5.3 6 1.7 2–18

0.60
1.87
1.12
Dec

Nuclear vacuole 0.6 0.3 0–3

70
60
71
85
74

6
8.2

Nuclear crest 0.1 6 0.1 0–1

Macrocephalic 0.4 6 0.3 0–2
Microcephalic 0.1 6 0.1 0–1
Proximal droplet 3.7 6 1.0 0–10
Concentration (3 109/ml)

Normal morphology (%)

Pseudodroplet 0.1 6 0.1 0–1

Progressive motility (%)
Viable (live) sperm (%)

Bowed midpiece 11.6 6 3.9 1–34

Intact acrosome (%)
Total sperm (3 109)

Reflexed distal midpiece 2.4 6 0.6 0–6

Total motility (%)

Bent tail 9.3 6 1.1 4–14

February 2011.

Coiled tail 0.2 6 0.2 0–2

Volume (ml)

Double tail 0.1 6 0.1 0–1

Short tail 0.1 6 0.1 0–1
Color

a
Samples with urine contamination or gel agglutination were
pH

excluded from this table.

 BEZJIAN ET AL.—MARKHOR GOAT SEMEN AND REPRODUCTIVE SEASONALITY
Figure 3. Photomicrograph of markhor sperm (31,000 magnification). A. Eosin–nigrosin stain of viable (live)
sperm with a bent tail. B. Eosin–nigrosin stain of viable (live) sperm with a proximal droplet (top) and a dead
sperm with a detached head (bottom). C. Spermac stain of two normal sperm with intact acrosomes (bottom left)
and a reacted acrosome (top right).
December were initially processed using slightly
different laboratory methods, so these samples
were excluded from further analysis. None of the
raw semen samples from February met the
inclusion criteria. Therefore, five semen samples
from four bucks were used for evaluation of
extender and processing method. The raw sam-
ples had the following characteristics: TM (68.0 6
6.8%), PM (57.0 6 6.4%), viable (live) sperm (49.0
6 7.4%), normal morphology (72.8 6 4.3%), and
intact acrosomes (64.0 6 3.4%).
Sperm motility, viability, acrosome integrity,
and normal morphology in individual extenders at
different time points were presented in Table 4.
The effect of processing method (centrifuged or
noncentrifuged) on semen parameters was not
significant alone or in interactions with time and
extender, so processing method was eliminated
from the statistical model. There were individual
effects of extender and time for all semen
parameters, except morphology; time had no
effect on morphology. Interactions between time
and extender were only significant for TM.
Therefore, no superscripts indicating differences
in sperm PM, viability, morphology, and acro-
some integrity in extenders at different time
points were presented in Table 4 as they would
have been generated by forcing two-way interac-
tions between extender and time back into the
statistical model. The overall effects of extender
and time on semen parameters, which took into
account only significant interactions, are detailed
as follows in the text.
For TM, there was an interaction between time
and extender (P , 0.005). Postequilibration TM
was higher for sperm in Tris–15% egg yolk
679
compared with sperm in Bioxcell and Tris–5%
egg yolk. Postthaw TM was similar for sperm in
Tris–5% and Tris–15% egg yolk, whereas sperm in
Bioxcell displayed significantly lower TM at 0, 3,
and 6 hr postthaw. At 9 and 12 hr postthaw, the
TM of sperm in Tris–5% egg yolk was still higher
than Bioxcell, but the TM of sperm in Tris–15%
egg yolk did not differ significantly from Bioxcell
or Tris–5% egg yolk. For PM, there were no
significant two-way interactions between extender
and time. Overall in the statistical model, there
were no differences in PM between sperm in Tris–
5% egg yolk and Tris–15% egg yolk (P . 0.10), but
sperm in both Tris–egg yolk extenders displayed
higher PM than sperm in Bioxcell (P , 0.05).
For viability, morphology, and acrosome integ-
rity, there were no significant two-way interac-
tions between extender and time. Overall, sperm
in Bioxcell displayed a higher percentage of viable
sperm than in Tris–5% and Tris–15% egg yolk (P
, 0.01). For morphology, there was a tendency (P
, 0.10) for an extender effect; sperm in Tris–5%
egg yolk tended to have a higher percentage of
morphologically normal sperm compared with
Bioxcell. The percentage of morphologically nor-
mal sperm in Tris–15% egg yolk did not differ
significantly from the other two extenders. The
percentage of sperm with normal acrosomes was
significantly higher in Bioxcell than in Tris–15%
egg yolk (P , 0.05), and tended to be higher in
Bioxcell than in Tris–5% egg yolk (P , 0.10).
DISCUSSION
The markhor bucks in this study displayed
reproductive seasonality with scrotal circumfer-
ence and testosterone inversely associated with
680 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Table 4. Sperm total motility (TM) and progressive motility (PM), with postthaw 0- and 3-hr evaluation of
sperm viability, normal morphology, and acrosome integrity. Semen samples (n ¼ 5) were collected by
electroejaculation of four markhor bucks and extended in Bioxcell, Tris–5% egg yolk, or Tris–15% egg yolk.
Values are means 6 SEM.a

Bioxcell Tris–5% egg yolk Tris–15% egg yolk

Postequilibration
TM% 41.5 6 3.9A 42.0 6 5.2A 53.5 6 5.3B
PM% 29.3 6 3.2 25.0 6 6.1 38.0 6 5.7
Postthaw 0 hr
TM% 25.6 6 7.7A 41.5 6 5.6B 41.5 6 5.5B
PM% 21.4 6 6.7 30.0 6 5.8 30.1 6 5.3
Viable (live) % 32.6 6 4.2 25.3 6 4.1 21.6 6 2.7
Normal morphology % 53.6 6 7.2 61.4 6 3.4 56.2 6 3.3
Intact acrosome % 47.0 6 5.4 26.0 6 7.8 29.5 6 7.9
Postthaw 3 hr
TM% 10.5 6 3.4A 36.5 6 3.7B 44.0 6 4.6B
PM% 7.9 6 2.7 11.0 6 1.3 17.5 6 3.0
Viable (live) % 22.8 6 2.2 14.5 6 3.9 15.2 6 3.1
Normal morphology % 45.1 6 6.5 52.9 6 3.5 48.3 6 2.9
Intact acrosome % 18.6 6 5.2 15.5 6 6.9 8.0 6 3.3
Postthaw 6 hr
TM% 3.0 6 1.2A 30.2 6 5.6B 26.3 6 4.9B
PM% 1.0 6 0.5 10.4 6 2.0 10.8 6 2.4
Postthaw 9 hr
TM% 1.0 6 0.8A 20.1 6 4.7B 10.2 6 3.2AB
PM% 0.2 6 0.1 6.3 6 1.4 3.8 6 1.3
Postthaw 12 hr
TM% 0.1 6 0.1A 13.6 6 5.5B 9.0 6 3.0AB
PM% 0.0 6 0.0 4.6 6 1.5 1.7 6 0.7
a
Different superscript uppercase letters in the same row denote significant differences (P , 0.05).

photoperiod, similarly to other short day length
breeders. In Syracuse, day length started to
decrease in June, with a rapid decline in the
amount of daylight by approximately 1.4 hr/mo
from August to November. During these months,
scrotal circumference, serum testosterone, and
fecal testosterone began to increase to their peak
values in November, when day length was short.
The significant positive association between se-
rum and fecal testosterone suggests that fecal
testosterone is an acceptable, noninvasive method
for monitoring circulating testosterone in mar-
khor bucks.
In the wild, the markhor breeding season
extends from November through December.5,52
In some ruminants, testosterone secretion in-
creases 50–60 days before the breeding season,
allowing time for a complete spermatogenic cycle
and progression of sperm through the epididymi-
des.18 Optimal sperm production and function
then coincide with female receptivity during the
breeding season. It has been previously hypothe-
sized that it is best to freeze electroejaculated
ruminant semen during the breeding season, when
sperm production is high and testosterone secre-
tion is actually decreasing, as testosterone may
modify sperm membrane fluidity, rendering
sperm more susceptible to damage during the
freeze-thaw process.10 In conjunction with this
study’s findings, this hypothesis suggests that
December and January are the optimal months
for markhor semen collection and cryopreserva-
tion. For comparison, future studies should
include semen collection and evaluation during
peak testosterone production in November, as
well as throughout the year.
Markhor semen collected in December and
January had similar total sperm numbers, but
the samples in December had higher sperm
motility, viability, and acrosome integrity. During
these months, markhor mean semen volume and
sperm concentration were comparable to those
reported for domestic goats collected by electro-
ejaculation, but total motility and normal mor-
phology were slightly lower those reported for
domestic goats.19,32 Characteristics of markhor
semen collected in December and January were,
however, consistent with those reported for other
 BEZJIAN ET AL.—MARKHOR GOAT SEMEN AND REPRODUCTIVE SEASONALITY
endangered, wild goats collected via electroejacu-
lation.26,52 In contrast to the previous 2 mo, semen
samples collected in February were of poor
quality, with low sperm number, motility, acro-
some integrity, and normal morphology. Urine
contamination and gel agglutination also were
detected during this month. Many confirmed
seasonal breeders show increased production of
functionally intact and morphologically normal
sperm during the breeding season, whereas others
exhibit aspermatogenesis outside the breeding
season.9,10,35,37 In this study, the poor sperm
quantity, motility, and morphology in samples
obtained during February suggest that markhor
bucks may follow a pattern of reduced ejaculate
quality toward the end of the breeding season. In
North American zoos, most observed markhor
births have occurred during May and June,
suggesting breeding dates from November
through January; however, fertile markhor breed-
ings have been documented as late as April, with
birth as late as October (LaBarge, pers. comm.).
Therefore, markhor semen collection and evalu-
ation throughout the year are needed for confir-
mation of the seasonality of ejaculate quality.
In addition to season, ejaculate quality may
have been affected by the collection method.
Electroejaculation typically yields a greater vol-
ume of semen with a lower concentration of
sperm than collection using an artificial vagina
(AV).19,32,65 The greater semen volume may be
attributed to urine contamination and increased
seminal fluid production from electrical overstim-
ulation of pelvic nerves and accessory sex
glands.7,32,65 Ideally, captive bucks would be
trained at a young age for semen collection by
using a teaser mount and an AV to eliminate the
need for general anesthesia and electroejacula-
tion, increase the potential frequency of semen
collections, and yield better quality semen sam-
ples. Markhor goats have extremely tall and
powerful horns, so the training process for captive
animals should only be attempted with extreme
caution and prioritization of handler and animal
safety. Because semen collection from most wild
species living in their natural habitats or in
captivity requires anesthesia and electroejacula-
tion, methods that maximize the fertility of even
poor-quality semen samples should be pursued.
Such methods should first include refinement of
collection, extension, and processing techniques.
Secondary methods could include application of
ARTs that use low numbers of sperm or even
compromised sperm.
681
Extender selection can be a complex issue when
dealing with caprine species, and attempts to
mimic basic ruminant techniques have yielded
mixed results. Secretions from the buck bulbo-
urethral gland are thought to have antimicrobial
or antioxidant properties, but they also contain
lipase-related proteins that coagulate egg yolk
proteins in extenders and produce sperm-toxic
substances.54,56,57 Although the precise protective
action of egg yolk during semen freezing is
unknown, it has been proven to be beneficial
during cryopreservation.21,57 Conventional meth-
ods for overcoming the harmful interactions of
seminal plasma and egg yolk have included the
use of low concentrations of egg yolk or dilution
of semen with extender, also known as coating the
ejaculate with extender, and then centrifugation to
pellet sperm and remove the seminal plasma
containing the lipases.10,29,31,49,57,66 Reducing the
time period of exposure to fluids from the
accessory sex glands has been shown to decrease
the harmful effects of seminal plasma on ejacu-
lated spermatozoa.63 Studies using various wash-
ing solutions, which do not contain egg yolk, for
the centrifugation process also have been evalu-
ated for caprine species.9,10,57 This extra processing
step of centrifugation requires time and equip-
ment, and it carries the risk of sperm loss and
damage.6,10,31,49,54 Results from this study did not
reveal a statistically significant difference between
centrifuged and noncentrifuged samples of post-
thaw markhor sperm. Therefore, omission of
centrifugation from the processing of markhor
semen with the extenders tested is recommended.
In this study, characteristics of semen extended
in Tris with the higher (15%) concentration of egg
yolk did not differ from the lower (5%) concen-
tration. This finding is unique when compared
with previous studies that show better mainte-
nance of sperm structural integrity in low egg yolk
concentration during the freeze-thaw process.10,54
Compared with the soy-based Bioxcell, sperm
cryopreserved in Tris with both concentrations of
egg yolk demonstrated greater TM at 0, 3, and 6
hr postthaw (P , 0.05) and greater overall PM (P
, 0.05). Although sperm motility appeared to
benefit from the protective action of Tris–egg yolk
during cryopreservation, viability and acrosome
integrity may have been compromised. Sperm
extended in Bioxcell displayed higher overall
viability than sperm in both Tris–egg yolk extend-
ers (P , 0.01), significantly more intact acrosomes
than sperm in Tris–15% egg yolk (P , 0.05), and a
tendency for more intact acrosomes than sperm in
Tris–5% egg yolk (P , 0.10). If motile sperm have
682 JOURNAL OF ZOO AND WILDLIFE MEDICINE
damaged acrosomes or if sperm with intact
acrosomes are unable to move through the female
reproductive tract, fertilization will not occur.
Therefore, breeding trials are needed to deter-
mine whether motility or acrosome integrity is
better correlated with sample fertility for markhor
goats.22 When comparing sperm motility and
viability in both raw and thawed semen samples,
motility was often greater than the percentage of
viable sperm determined by identifying cells that
excluded eosin stain. The reason for this discrep-
ancy is not readily apparent as it has not been
encountered by us during routine semen evalua-
tion of domestic small ruminants. Accordingly, in
future markhor studies, comparison of different
methods of viability, such as the hypoosmotic
viability test, fluorescence staining, or flow cy-
tometry, is recommended.
For shipment of semen, extenders that do not
contain ingredients of animal origin are preferred
for biosecurity and consistency. Although the
postthaw motilities of markhor sperm were not
as high with Bioxcell as they were with the Tris–
egg yolk-based extenders, advanced processing
methods for thawed semen can still promote
successful fertilization. In domestic species, the
motility of frozen-thawed sperm typically de-
creases by 40–50% compared with fresh sperm;
however, using insemination doses containing
larger quantities of sperm can sometimes com-
pensate for the loss of motility due to cryopres-
ervation.3 Noncompensable factors, such as
individual buck sperm competency to complete
fertilization or sustain embryogenesis, are diffi-
cult to overcome and can be evaluated with
breeding soundness examinations in conjunction
with kidding rates.53 In addition, advanced pro-
cessing techniques such as washing, migration,
filtration, and colloid centrifugation methods can
be used to ensure selection of good-quality
spermatozoa before use in ARTs. Centrifugation
through a colloid may offer the best possibility for
selecting good quality sperm and for removing
cellular debris and pathogens that may be present
in seminal plasma.38 Postthaw protocols for
Norwegian dairy goats predict a kidding rate of
57–62% by using samples with TM of at least 50%
with a minimum concentration of 400 3 106
sperm/ml, and a minimum volume of 0.5 ml for
vaginal insemination during natural estrus.40 In
domestic goats, pregnancy rates from 35 to 65%
can be achieved using a total of 180 3 106 frozen-
thawed sperm for cervical insemination or as little
as 5–20 3 106 motile sperm for laparoscopic
intrauterine insemination.15,50 Semen samples that
may not be of high enough quality for cervical or
intrauterine insemination may still be used for in
vitro fertilization or intracytoplasmic sperm in-
jection.
CONCLUSIONS
In summary, markhor bucks demonstrated
reproductive seasonality, with scrotal circumfer-
ence and testosterone inversely associated with
day length. Maximum values for scrotal circum-
ference, serum testosterone, and fecal testoster-
one occurred in November. Moderate-quality
semen samples were collected by electroejacula-
tion in December and January, whereas poor-
quality samples were collected in February. No
significant differences in postthaw semen charac-
teristics were detected between centrifuged and
noncentrifuged samples, suggesting that this step
may be eliminated from the processing of mar-
khor semen with the extenders tested. Sperm
cryopreserved in Tris-based extender with 5 and
15% egg yolk displayed greater motility postthaw
than sperm in Bioxcell. Sperm in Bioxcell dis-
played greater postthaw viability and had more
intact acrosomes than sperm in Tris–egg yolk
extenders. Further studies should include sam-
pling a larger cohort of markhor, semen collection
and evaluation throughout the year, and insemi-
nation trials to correlate postthaw sperm charac-
teristics with sample fertility.
Acknowledgments: The authors are profoundly
grateful to the following people for help during
this research: Mr. Thomas LaBarge and the staff
of the Rosamond Gifford Zoo for dedication to
this study and Mrs. Sue Faso for excellent clinical
assistance during sample collection at the Zoo;
Mr. Jason Barry at the Cornell Statistical Con-
sulting Unit for statistical assistance; and Ms.
Nicole Presley, Ms. Nicole Parker, and Dr. Janine
Brown at the Smithsonian Conservation Biology
Institute for testosterone analysis. Funding was
provided by the Clinical Research Grant Pro-
gram, Cornell University, College of Veterinary
Medicine, Ithaca, New York 14853, USA.
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Received for publication 10 November 2012