The application of reproductive technologies to natural populations of red deer.
ABSTRACT Over the past decade, there has been increasing interest in the application of reproductive technology to the conservation and management of natural populations of deer. The application of assisted reproduction technologies within natural population of deer is in its infancy. However, its future potential is enormous, particularly in relation to genetic management or conservation. This paper reviews the present state of such technologies for a wild subspecies of red deer, the Iberian red deer (Cervus elaphus hispanicus), by discussing the major components of oestrous synchronization, semen collection/cryopreservation and insemination techniques. In addition, findings made during the course of studies on natural populations have enormous potential for the understanding of novel reproductive mechanism that may not be uncovered by livestock or human studies. A summary of these results are also reviewed here.
- SourceAvailable from: William Pérez[show abstract] [hide abstract]
ABSTRACT: To describe the macroscopic anatomy of the genital organs of the male pampas deer (Ozotoceros bezoarticus), organs from ten animals found dead in a captive breeding station were dissected. The unpigmented scrotum was located in the inguinal region near the body, and was covered by the thighs. In the investments of the testicles the dartos tunic was greatly developed. The cremaster muscle was located dorsocaudal to the testicle, and was divided caudally into three bundles of fibers. The right testicle was significantly heavier than the left, and there was a positive relationship between body weight and the weight of both testicles. The tail of the epididymis, ventrally located, had a caudal portion attached to the caudal extremity of the testicles by the proper ligament of the testicles, and a portion elongated free caudally located. The deferent duct was located caudomedially to the corresponding testicle. The accessory genital glands were the ampullary glands, vesicular glands, and a small pars disseminata of the prostate. The penis was fibroelastic, without sigmoid flexure, with a thick albuginea. The retractor penis muscle was very long, and ended in the distal part of the penis near the rudimentary glans. The general disposition of the male genital organs of the pampas deer were similar to that of other ruminants, with some differences, such as size and location of the testicles, the absence of the sigmoid flexure of the penis, and fewer accessory genital glands.Anatomical science international. 02/2013;
- [show abstract] [hide abstract]
ABSTRACT: Small ruminants are an important component of the global production systems of meat and wool, and their reproductive biology is well known. However, the incorporation of assisted reproduction techniques (ART) in the production systems of small ruminants is not as well developed as for other domestic species. Normally, production systems that incorporate ARTs are restricted to artificial insemination or in vivo embryo transfer. Intracytoplasmic sperm injection (ICSI) is one of the ARTs techniques reported for small ruminants and consists of the injection of spermatozoa inside an oocyte, bypassing the natural process of sperm-oocyte interaction. In goats and sheep, there are few live births by ICSI reported, with no reports from other species of small ruminants. Currently, there has not been intensive research about ICSI in small ruminants. However, ICSI has potentially important applications in animal production systems, primarily its use with semen of valued animals, with epididymal sperm, in the fertilization of prepubertal or cryopreserved oocytes. Other applications include more advanced techniques, such as transgenic-ICSI or its combination with spermatogonial transplantation. In this article, we review the "state of the art" of this technique in small ruminants including its historical development, research needs for its improvement and future applications.Animal reproduction science 07/2012; 133(3-4):129-38. · 1.56 Impact Factor
- Anantomia Histologia Embryologia 01/2013; 42(3):168-174. · 0.88 Impact Factor
The application of reproductive technologies to natural populations of red deer
J. Julián Garde1, F. Martínez-Pastor1, M. Gomendio2, AF. Malo2, Ana J. Soler1, MR.
Fernández-Santos1, MC. Esteso1, L. Anel3, ERS. Roldán2.
Abridged title: Red Deer and Reproductive Technologies
1Reproductive Biology Group, Instituto de Investigación en Recursos Cinegéticos, IREC,
(UCLM-CSIC-JCCM). Campus Universitario sn, 02071. Albacete, Spain
2Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC),
3Animal Reproduction and Obstetrics, University of León, 24071, León, Spain
Over the past decade, there has been increasing interest in the application of reproductive
technology to the conservation and management of natural populations of deer. The
application of ART within natural population of deer is in its infancy. However, its future
potential is enormous, particularly in relation to genetic management or conservation. This
paper reviews the present state of such technologies for a wild subspecies of red deer, the
Iberian red deer, by discussing the major components of oestrous synchronization, semen
collection/cryopreservation and insemination techniques. In addition, findings made during
the course of natural populations studies have enormous potential for the understanding of
novel reproductive mechanism that may not explain during livestock animal or human
studies. Finally, the results of these studies are also reviewed here.
Rapid expansion of the red deer (Cervus elaphus) farming industry around the world within
the last 35 years has been accompanied by equally rapid development and adoption of a
number of assisted reproduction technologies (Asher et al. 2000). These assisted reproduction
technologies (ART) have not only facilitated increased rates of genetic improvement on
individual farms, but have also allowed widespread movement of genetic material around the
world, have been implicated in genetic rescue of rare genotypes/individuals, and have allowed
farmers and researches to cross species boundaries in the production of potentially useful
hybrids (Asher 1998; Asher et al. 2000). The principle tools that have been used, within the
red deer farming, include artificial insemination (AI), multiple ovulation embryo-transfer
(MOET) and in vitro embryo production (IVP).
If one excludes reindeer, red deer and their hybrids with wapiti/elk are by far the most
widely farmed species. In New Zealand, there are now around 2.6 million farmed deer, of
which 85% are red deer and the balance hybrid red deer (Fletcher 2001); there are also small
numbers of farmed fallow deer. During 2000, this industry exported deer products worth $81
million US, of which venison contributed $60 million and velvet $11 million; the balance was
made up of hides and other co-products (Fletcher 2001). Demand for New Zealand farmed
venison was exceptionally high in Europe in 2001 and the New Zealand deer farmer
experienced prices 35% higher than at the same time in 2000, and 63% higher than in 1999
(Fletcher 2001). These figures are especially impressive given that this industry has been
developed entirely in the last 35 years in a nation of only 3.5 million people, and receiving no
agricultural support. Other national farmed deer herds are much smaller than New Zealand’s.
With such a rapid build up of farmed deer herds, there has been economic pressure for
deer breeders to purchase specific bloodlines based on heavily promoted, phenotypically
high-performing individual animals. By the mid 1980s, artificially insemination with
cryopreserved semen was commercially available to New Zealand deer farmers (Asher et al.
2000). The efficiency of laparoscopic intrauterine insemination in red deer, assessed as
conception rate (pregnancies established per 100 hinds inseminated), generally ranges from
55% to 70% for fresh and frozen-thawed semen, respectively (Asher et al. 2000).
Conservative estimates of AI practice in New Zealand indicates insemination of 10000 red
deer hinds annually, representing only 1–2% of breeding hinds (Asher 1998). This percentage
has not changed significantly, although the recent development of transcervical insemination
(Willard et al. 2002), and improved genetic evaluation, especially through the development of
sire-referencing, could lead to more widespread use.
In recent years, there has been increasing interest in the application of reproductive
technology to the conservation and management of natural populations of deer (Jabbour and
Bainbridge 1997). The application of ART within natural population of deer is in its infancy.
However, its future potential is enormous, particularly in relation to genetic management or
conservation, as in the case of the Iberian red deer (Cervus elaphus hispanicus). It is the
largest free-living ruminant of the Iberian peninsula, which lives remarkably southern than the
much more studied Scottish red deer (C. elaphus scoticus). This subspecies differs from C.
elaphus sp. mainly because it is smaller in size. ART may play an important role for the
purpose of ensuring genetic preservation and/or genetic progress, which are becoming
increasingly important as a result of the genetic isolation of wild populations within fenced
games estates (Martinez et al. 2002). Deleterious effects of inbreeding have been found on
some components of the fitness of hinds (Coulson et al. 1998) and also on male reproductive
function in others ungulate species (Roldán et al. 1998; Gomendio et al. 2000). In this
situation, germplasm conservation of Iberian red deer offers the possibility of genetic
variability preservation via biotechnological reproduction programmes. In addition, the only
commercial benefit obtained from this subspecies is that of hunting. The genetic value of the
Iberian red deer population is well known worldwide, and the conservation of this genetic
potential is an important incentive for the Spanish game breeding and hunting industry.
Besides, there is also a remarkable interest in the use of ART for genetic salvage, particularly
from harvested trophy males (Asher et al. 2000). Unfortunately, Iberian red deer is the carrier
of tuberculosis (Gortazar et al. 2005) and theileriosis (Höfle et al. 2004)). This subspecies
might therefore not be translocated from one part of Spain to any other area, due to the
potential of spreading these diseases (Fernandez de Mera et al. 2003). ART thus represent
methods to breed disease-free Iberian deer and may have economic potential for game
farming in the future.
The reasons stated above highlight the importance that ART may have in managing
deer natural populations, as is the case of Iberian red deer. Of the genetic material in cryo-
banks, the collection, storage and subsequent use of spermatozoa has found the most wide-
spread application (Watson and Holt 2001). According to this, cryopreservation of
spermatozoa combined with artificial insemination (AI) has been the method of ART that has
been most extensively applied to deer species (Asher et al. 2000). In our context, post-mortem
recovery of spermatozoa from the epididymides of hunted stags has been proposed as a
widely used source (Platz et al. 1982), since collection by other methods is often very difficult
or unaffordable. Also, if the species are hunted, a considerable number of samples may be
available each year, which can eventually be used to restore populations challenged by
inbreeding and to reinvest the valuable genetic material in the population (Platz et al. 1982).
Thus, this paper reviews the present state of such technologies for a wild subspecies of
red deer, the Iberian red deer, by discussing the major components of oestrous
synchronization, semen collection/cryopreservation and insemination techniques. In addition,
findings made during the course of natural populations studies have enormous potential for
the understanding of novel reproductive mechanism that may not explain during livestock
animal or human studies. Finally, the results of these studies are also reviewed here.
Iberian red deer hinds are seasonally polyestrous (García et al. 2002), and exhibit an oestrous
cycle varying from 19 to 20 days (García et al. 2003a). This subspecies of red deer is a short-
day breeder that mates during a rut occurring shortly after the autumn equinox (García et al.
2002). Indeed, it is well known that mammalian species from temperate areas trigger their
reproductive physiology in response to environmental cues. It has long been accepted that the
photoperiod is the primary environmental cue controlling seasonal breeding in deer. Thus, the
photoperiodic signal is transduced by the pineal gland into a pattern of melatonin secretion
which, in turn, provides a critical endocrine signal to regulate secretion of other hormones
involved in the onset and end of the annual breeding season. We found for first time in red
deer that the pineal gland of the adult female is highly responsive to both daily and seasonal
changes in natural environmental illumination, although overnight levels lasted longer than
the photoperiodic night in all cases, particularly at the winter solstice (García et al. 2003b).
In these species where females exhibit regular cycles of sexual activity,
synchronization of oestrous and ovulation is essential, to allow assisted breeding procedures
to be applied at a time when the chances of conception are maximal. In most species of deer,
recurrent ovulation occurs as a direct consequence of failed conception during the previous
cycle (García et al. 2003a). The progesterone-secreting corpus luteum is destroyed by the
action of protaglandins secreted by the non-pregnant uterus, and this precipitates a subsequent
cycle of oestrous activity and ovulation. There are a number of practical advantages to
exerting external control over timing of reproductive events, particularly in terms of
improving efficiency of artificial insemination. In addition, natural (spontaneous) oestrous is
difficult to detect. Furthermore, natural synchrony is no better than 10-14 days, been
necessary successive AI of hinds over this period, a situation which is clearly impractical
(Asher 1998). Therefore, oestrous synchronization of red deer hinds is an important facet of
all AI programmes of this species.
Synchronization of oestrous and ovulation in a group of females can be induced
artificially by altering the endogenous endocrine environment of a reproductive active, non-
pregnant female through the exogenous administration of prostaglandins or progesterone
(Asher et al. 1993). A large number of studies have investigated the use of the controlled
internal drug releasing (CIDR) device, containing progesterone, for efficacy control of oestrous
in red deer. Besides, the additional administration of equine chorionic gonadotrophin (eCG) at or
near of CIDR device withdrawal is generally performed for farmed red deer. For more details
see Asher et al. (1993). This procedure has also proven successful for Iberian deer by our group
(García et al. 1998). Thus, we have adopted this method for artificial synchronization of
oestrous as a more cost-effective alternative to detection of natural oestrous (Soler et al.
2003b; Malo et al. 2005a). The use of prostaglandin injections to synchronize hinds has been
investigate, but has generally proved to be of lower efficacy than intravaginal CIDR (Asher et
al. 1993). It should be borne in mind, however, that the optimum timing of insemination in
relation to the time of oestrous synchronization treatment varies between species and is
dependent on the subtle interplay between the hormones oestrogen (secreted by the
developing follicle) and LH (secreted by the pituitary), which leads to follicle rupture and
release of the egg. The aim must be deposit the spermatozoa at a time close to ovulation, so
that only one spermatozoon will penetrate the ovum, leading to successful fertilization and
subsequent embryonic development.
Semen collection and quality
Farming of red deer have allowed to apply conventional artificial reproduction techniques for
obtaining semen from stags, using either artificial vagina or electroejaculation. However, in
the context of natural populations, the use of techniques based in collection by artificial
vagina must be discarded, although it has been successful with tamed animals (Gordon 1997;
Gizejewski 2000, 2004). To the contrary, electroejaculation can be used combined with
darting, thus it is feasible to apply it to wild animals. Nevertheless, the most practical
technique is post-mortem recovery of epididymal spermatozoa, since this species is legally
hunted in many countries. These techniques have been tested not only in red deer, but also in
many other wild ruminant species (Asher et al. 2000; Holt 2001). Despite of the many
published studies dealing with deer semen, few of them give enough detail on the method
used for seminal collection, techniques vary and it is difficult to compare different studies.
Electroejaculation must be used when the objective is either to obtain a representative
number of samples from natural populations not subjected to hunting, or to repeatedly recover
samples from selected males. To achieve a viable methodology, not only a suitable
electroejaculation technique must be developed, but also effective and secure darting and
anesthesia. Reports on electroejaculation of red deer stags are referred from more than 30
years ago (Jaczewski and Jasiorowski 1974). The major problems related to this techniques
are related to the possible risks of the anesthetic protocol (Krzywinski and Wierzchos 1992),
and to the quality and quantity of the semen sample, since dead of valuable individuals
because of anesthesia or bad quality or insufficient semen sample can make a collection plan
unworthy. Indeed, electroejaculated semen has a different composition that semen collected
by "natural" methods (artificial vagina), because of differential stimulation of seminal glands
by the electroejaculation probe and the difficulty of achieve repeatability between
electroejaculation sessions. This may negatively affect semen quality, but it is still to be
confirmed for red deer (Asher et al. 1993, 2000).
Several authors have reported successful electroejaculation with restrained males
(Haigh et al. 1984b; Comizzoli et al. 2001b), which may not be applicable due to animal
welfare concerns. Regarding anesthetic protocols, successful collection has been achieved
using fentanil citrate, xylacine or azaperone (Fennessy et al. 1990). Sipko et al. (1997)
reported the use of diacethylcholine+displacine for Siberian Maral (C. e. sibiricus). Our group
has reported xylacine+ketamine as an effective and secure preparation for Iberian red deer (C.
e. hispanicus) anesthesia prior to electroejaculation, with yohimbine as inhibitor (Anel et al.
2000; Garde et al. 2000; Martínez et al. 2004; Martínez-Pastor et al. 2004, 2006). We have
carried out 192 electroejaculations on 25 red deer stags with no fatal incidents, recommending
intubation and monitoring of the animals during anesthesia (Anel et al., unpublished data).
Regarding the electroejaculation protocol, few articles give details on probe
characteristics, electroejaculation process or the applied current. Probe size and characteristics
are described in few reports (Haigh et al. 1984b; Fennessy et al. 1990). In our experiments
with Iberian red deer, we used a 25030 mm probe with three longitudinal electrodes
(Martínez et al. 2004; Martínez-Pastor et al. 2004, 2006), achieving ejaculation at average
voltage and amperage values of 4.5 V and 90 mAmp, respectively. According to Sipko et al.
(1997), successful electroejaculation has been achieved in Siberian Maral using 3–4
stimulations of 5 s each, at 10 s intervals, with 8 V as maximum.
Samples obtained by electroejaculation are suitable for preparation of seminal doses
and cryopreservation. However, electroejaculation method, individual and season influences
the outcome (Fennessy et al. 1990; Sipko et al. 1997). However, electroejaculation can
stimulate urine emission, which may spoil good quality samples and contribute to sample
variability. Regarding this issue, we have reported that 22 samples of 79 obtained by
electroejaculation from Iberian red deer were contaminated with urine (Martinez-Pastor et al.
Seasonality is a major factor conditioning sample collection in red deer, not only
quality, but also quantity. Haigh et al. (1984a) could obtain seminal doses during the breeding
season of wapiti, but no semen was obtained in June and July. Anel et al. (2000) studied the
quality and quantity of semen collected by electroejaculation in autumn, winter and spring
(after antler casting), finding that semen concentration and volume reached its highest values
in autumn, and decreased in winter to a minimum in spring. However, quality was similar in
autumn and winter, only dropping in spring. Other characteristics of the ejaculate vary too
(Gizejewski et al. 2003; Gizejewski 2004). For instance, during the rut, semen is accompanied
by a sticky and viscous yellow secretion of the vesicular glands, termed “honey”, whose
presence may affect spermatozoa quality. Anel et al. (2000) reported presence of this secretion
when electroejaculating in autumn, but absence in winter.
Post-mortem seminal recovery is the most practical option to obtain spermatozoa
samples from wild populations of red deer. Besides, hunting provides a constant source from
harvested animals. The source of post-mortem spermatozoa is the cauda epididymis. The
maturation stage and fertility of spermatozoa stored in this part of the epididymis are similar
to those of ejaculated spermatozoa and, therefore, they are suitable for germplasm banking
(Foote 2000). Sperm recovery is usually carried out either by performing some cuts on the
cauda epididymis (Anel et al. 2002; Garde et al. 2000; Gizejewski et al. 1998; Martínez-
Pastor et al. 2002, 2006) or by retrograde flushing of the cauda from the vas deferens
(Comizzoli et al. 2001a; Esteso et al. 2003; Fernández-Santos et al. 2006a,c; Garde et al.
1998a, 1998b; Soler et al. 2003b, 2005a, 2005b; Zomborszky et al. 1999). Recovery by means
of cuts is quicker and more easily performed than retrograde flushing, but it has the important
drawback of contamination, negatively affecting the spermatozoa. We have compared both
methods (Martinez-Pastor et al. 2006), finding that the total amount of spermatozoa recovered
was equivalent. Sperm quality, both before and after cryopreservation, was slightly better
when retrograde flushing was used, and sample contamination was clearly higher for the cuts
method. In fact, the median values of the concentration of erythrocytes for cuts and flushing
were around 710 and 10 mL, respectively. The presence of such contaminants in the sample
may be deleterious, thus, in routine recovery for germplasm banking it would be preferable
the use of methods which minimize contamination.
As in the case of electroejaculation, many factors affect semen quality, and, apart from
the technique of sperm recovery and factors intrinsic to the male, season is the most
significant one (Guerra et al. 2002). Our group (Martinez-Pastor et al. 2005d) showed that the
number of recovered spermatozoa varied dramatically from a median value of 35.110 mL
during the rut (September to mid-October) to 13.210 mL in the post-rut (mid-October to
December) and to 2.110 mL in February (non-breeding season). However, in the same
study, sperm quality was as good, and even higher (acrosomal status and sperm viability), in
the post-rut as in the rut. However, sperm recovered in February was barely motile. In another
study (Martinez-Pastor et al. 2005a), we found differences between samples collected during
the rut and during the post-rut, regarding motility subpopulations. These differences were
attributed to the maturational status of the epididymal spermatozoa, and its impact on sperm
fertility has not been established yet.
In the case of post-mortem samples, the time between the death of the animal and
sperm collection and processing is an important factor to take into account. Indeed, several
studies on Iberian red deer indicate that sperm samples undergo a quick drop in motility after
24 h post-mortem, although other characteristics are not affected but many days post-mortem
(Anel et al. 2002; Garde et al. 1998b; Martinez-Pastor et al. 2005a, 2005c; Soler and Garde
2003; Soler et al. 2005a). Therefore, epididymal samples must be recovered and
cryopreserved as soon as possible after the death of the stag. Nevertheless, a few hours of
delay (considering transportation from the field to the lab) may not represent a difference.
Another fundamental landmark in a semen recovery and cryopreservation program is
the assessment of the quality of the samples. Many conventional and novel techniques have
been successfully used for the assessment of red deer spermatozoa, and recently developed
techniques have been adapted to analyze red deer spermatozoa, increasing the amount of
information available. Thus, CASA systems have been used to analyze sperm motility
parameters (Malo et al. 2005b; Martinez-Pastor et al. 2005c, Martínez-Pastor et al. 2006b,c),
and it has been possible to identify subpopulations defined by motility patterns, and their
changes in different circumstances (Martínez-Pastor et al. 2005a, 2005b). Moreover,
fluorescent probes (either by fluorescence microscopy or by flow cytometry) have been used
to analyze different physiological parameters of red deer semen, as viability and acrosomal
status (Anel et al. 2002; Martínez-Pastor et al. 2006a, b, c), or mitochondrial membrane
potential (Martínez-Pastor et al. 2002; Soler et al. 2005a). Flow cytometric techniques for
DNA assessment (García-Macias et al. 2006; Soler et al. 2005a) and automated morphometric
analysis of sperm heads (Esteso et al. 2003; Soler et al. 2005b) have been adapted for its use
in this species.
Cryopreservation of red deer semen
The cryopreservation of the seminal doses is a fundamental step in the management of
germplasm banks, and one of the most critical ones. Spermatozoa must endure a series of
stresses, and semen samples undergo a more or less marked loss of viability and fertility.
Cryopreservation of red deer seminal samples has been reported for many years (Jaczewski et
al. 1976, 1978; Krzywinski 1981; Sipko et al. 1997a), and both refrigerated and frozen storage
are carried out in farmed red deer (Asher et al. 2000; Gordon 1997). However, there is a lack
of optimized protocols, especially for epididymal spermatozoa. Most protocols for
cryopreserving red deer sperm come from existing methods for ejaculated semen of domestic
ruminants, with little modifications.
Jaczewski et al. (1978) used protocols from bull, reindeer and goat for the
cryopreservation of red deer semen obtained by electroejaculation, and freezing the semen in
pellets on dry-ice. These authors obtained good post-thawing motility after using the goat
extender, although they could not determine if it was due to good extender choice or to the
different season of collection. Pellet freezing has been used by other authors (Krzywinski
1981; Sipko et al. 1997b), with good results. However, health standards and practical reasons
make preferable packaging in straws (Holt 2001), being 0.25 mL straws the preferred choice
for red deer.
Asher et al. (2000) reviewed thoroughly the storage and freezing of semen from many
species of deer. For red deer, they indicated that most authors have used protocols from sheep
and cattle with no modifications, but few studies had attempted to search new extenders for
red deer. Veldhuizen (1994) tried five extenders and three cryoprotectants (glycerol, dimethyl
sulfoxide and propan-1,2-diol). The results indicated that the Tris-citrate-egg yolk-glycerol
rendered the highest motility post-thawing, whereas lactose-egg yolk-glycerol better protected
acrosomes. However, in vivo fertility did not indicated differences. In any case, glycerol is the
preferred permeating cryoprotectant in deer. More recently, other authors have reported good
semen quality using commercial extenders for cattle (Zomborszky et al. 2005). Garde et al.
(2000) tried 4% and 8% glycerol concentrations on electroejaculated semen (Tes-Tris-
fructose-egg yolk extender), finding that 4% rendered slightly better results after thawing, but
not significant. In a recent report, we found that sperm viability after thawing was
significantly higher in samples frozen with 4% glycerol (Martínez-Pastor et al. 2006b). In the
same study, we compared three extenders with osmolalities of 320, 380 and 430 mOsm/kg,
finding higher post-thawing progressive motility and sperm viability with the 320 mOsm/kg
Nevertheless, epididymal spermatozoa have some peculiarities that make compulsory
to adapt or develop new extenders. These spermatozoa are in an environment totally different
from ejaculated ones. Epididymal fluid has a much higher osmolality than seminal plasma,
and its composition differs considerably regarding proteins and antioxidants. Therefore,
epididymal spermatozoa are not submitted to some interactions with seminal plasma
components, which may alter their capacity to reach and fertilize the oocyte. Despite these
facts, there are reports of successful use of cryopreserved epididymal spermatozoa for AI on
red deer after using extenders and protocols for cattle. Garde et al. (1998a) obtained four
fawns after vaginal AI of 17 synchronized hinds, using Triladyl® as semen extender. In
another experiment, our group tried three extenders: sodium citrate-fructose, Triladyl® and a
Tris-lactose extender (Garde et al. 1998c; Ortiz et al. 1997). We found that the lactose
extender (slightly hypertonic relative to the other two extenders) was the most suitable for
freezing high quality samples, but low quality samples were better preserved by Triladyl®.
Besides, Zomborszky et al. (1999) reported AI of superovulated hinds with epididymal
spermatozoa frozen in Tris-citric acid-fructose-egg yolk extender (two fractions; 6% final
glycerol concentration); uterus were flushed, recovering three embryos, which were
cryopreserved and two of them transplanted, given one fawn.
Recently, many studies have approached the development of optimized extenders and
protocols for red deer epididymal spermatozoa cryopreservation. Thus, Garde et al. (2000)
tried 4% and 8% glycerol for freezing epididymal spermatozoa in a Tes-Tris-fructose-egg
yolk extender, being 8% more effective protecting the membrane functionality as assessed
post-thawing. In another study, Martínez-Pastor et al. (2006b) confirmed this result, although
they only obtained statistically higher results regarding acrosomal status. Furthermore, in the
second part of their study on epididymal spermatozoa, Martínez-Pastor et al. (2006b) found
that 380 and 430 mOsm/kg extenders were more adequate than a 320 mOsm/kg extender for
freezing. These results differ from those corresponding to semen obtained by
electroejaculation, as showed above. Our results indicated that the mean osmolalities of
ejaculated and epididymal samples were 336 and 387 mOsm/kg, and therefore it would
influence the suitability of extender osmolality. This kind of studies point out the differences
between ejaculated and epididymal spermatozoa, and the need of treat them differentially
when designing cryopreservation protocols.
Besides, Fernández-Santos et al. (2005) tested the effect of glycerol, ethylene glycol,
propylene glycol and dimethyl sulfoxide, added at 22 °C or 5 °C. They found that dimethyl
sulfoxide was toxic to red deer epididymal spermatozoa, whereas glycerol performed slightly
better than ethylene glycol and propylene glycol. Regardless of the cryoprotectant used, they
found that adding it at 22 °C better protected the acrosomes. In two related studies, these
authors reported that 6% glycerol provided better protection than 4% glycerol. In the same
study, they studied if egg yolk concentration (0, 5, 10 and 20%) and its processing (clarified
by centrifugation or whole egg yolk) affected epididymal spermatozoa cryopreservation,
concluding that 20% clarified egg yolk improved sperm motility post-thawing. Other
experiment (Fernández-Santos et al. 2006a) agreed that 20% egg yolk is a more suitable
concentration for red deer epididymal spermatozoa than 0 or 5%, when cooling the semen
sample from 22 °C to 5 °C. In the same studies, we also analyzed the effect of different
velocities for cooling extended semen samples from 22 °C to 5° C. We found that a fast
velocity (4.2 °C/min vs. 0.23 °C/min) improved sperm motility after cooling and after
thawing. In other study (Martinez-Pastor et al., 2006c) we found that adding seminal plasma
from electroejaculated samples of red deer improved sperm quality before and after freezing.
Besides, Fernandez-Santos et al. (2006b) found that several antioxidants (specially catalase
and superoxide dismutase) improved freezing of red deer epidymal spermatozoa.
Complementarily, in other studies, we have dealt with the thawing procedures on
epididymal sperm cryopreservation. Thus, Soler et al. (2003a,b) studied the effect of different
thawing rates after freezing semen straws using liquid nitrogen vapor. We found that slow
thawing rates (thawing bath at 37 °C) rendered the best results, both for in vitro quality
assessment and for fertility (intrauterine AI; 69.7% for 37 °C/min vs. 42.4% for 70 °C/min).
Furthermore, morphometric assessment of sperm head dimensions after thawing showed that
the rapid thawing procedure (70 °C/min) caused a more marked head size decrease, and a
greater loss of sample heterogeneity, which was related to a higher degree of cryoinjury
(Esteso et al. 2003).
Finally, the most obvious result of our experiments was the clear demonstration of stag-
to-stag differences in response of their spermatozoa to freezing and thawing (Soler et al.
2003b; Esteso et al. 2006). In addition, we have previously reported that fertility rates also
vary markedly between males when frozen-thawed semen was used (Malo et al. 2005a).
Differences in the resistance to thawing of the spermatozoa of different individuals have been
observed for spermatozoa of other domestic (see Curry 2000) and wild (see Leibo and
Songsasen 2002) species. Within this context, semen donors have routinely been categorized
as “good” or “bad freezers”. Although similar experiences have been reported for several
species, no explanations for these differences have been substantiated. The mechanisms
underlying differences in cryosensitivity between different individuals have yet to be
elucidated, but there is some evidence for physiological differences between spermatozoa
from individuals of the same species (see Leibo and Bradley 1999). In this sense, we have
recently reported that differences in epididymal sperm-head area and shape exist between
“good” and “bad” freezers stags before freezing, with the smallest overall sperm head
dimensions found in the “good” freezers males (Esteso et al. 2006). Besides, we found that
the sperm heads in the fresh samples from the “good” freezers were more elongated and
narrow than those from the “bad” group (Esteso et al. 2006). In this sense, the sperm head
length in the fresh samples from “good” freezers was approximately two times higher than the
width. Taken together our results, we hypothesized that sperm head area and shape cause
differences in heat exchange as well as in movements of water and ions. It is, therefore,