Low temperature storage of rhesus monkey
spermatozoa and fertility evaluation by
Richard R. Yeomana,*, Shoukhrat Mitalipova,
Behzad Gerami-Nainib, Kevin D. Nusserc, Don P. Wolfa
aDivision of Reproductive Sciences, Oregon National Primate Research Center,
Oregon Health and Science University, 505 185th Avenue,
Beaverton, Oregon, OR 97006, USA
bWisconsin National Primate Research Center, Madison, WI 93706, USA
cDepartment of Otolaryngology, Oregon Health and Sciences University, Portland, OR 97201, USA
Received 25 March 2004; accepted 11 May 2004
The objective was to develop a sperm freezing procedure suitable for use in the propagation of
valuable founder animals by assisted reproductive technologies. Here, we report a comparison of
processing methods by measuring the motility of fresh and frozen–thawed rhesus monkey sperma-
tozoa and fertility via intracytoplasmic spermatozoa injection (ICSI) of sibling oocytes. Washed
spermatozoa were frozen in straws or in pellets using different cryoprotective media and processed
post-thaw with or without a density gradient centrifugation step. Among the four study series,
motility post-thaw was improved with density gradient centrifugation (17–24% versus 75%,
P < 0.01) achieving levels similar to fresh spermatozoa. Spermatozoa injected oocytes (total
n = 377) were co-cultured on BRL cells and observed for fertilization and development. With
spermatozoa frozen instrawsinliquidnitrogenvapors, the fertilization rate after ICSI was lower than
with fresh spermatozoa (40–44% versus 77–86%, P < 0.05), even with the Percoll-enriched fraction
that exhibited robust motility. In contrast, somewhat slower freezing of spermatozoa in pellets on dry
ice supported fertilization rates (73%) that were similar to the fresh counterpart. Developmental rates
of fertilized eggs were similar in all experiments. A total of 106 embryo transfers has resulted in the
first primate born after ICSI with F/T ejaculated spermatozoa plus 22 other infants to date.
Theriogenology xxx (2004) xxx–xxx
* Corresponding author. Tel.: +1 503 533 2489; fax: +1 503 690 5563.
E-mail address: email@example.com (R.R. Yeoman).
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
Additionally, a 3–4 h incubation after thawing improved the fertilization rate with spermatozoa from
a male with poor post-thaw recovery of sperm motility. In conclusion, an acceptable fertilization rate
after ICSI with motile, frozen–thawed primate spermatozoa was observed comparable to that
obtained with fresh spermatozoa allowing small quantities of competent spermatozoa to be used
with ICSI to facilitate propagation of desirable primate genotypes.
# 2004 Elsevier Inc. All rights reserved.
Keywords: Cryopreservation; Fertilization; ICSI; Rhesus monkey; Spermatozoa
The non-human primate is highly relevant as a human disease model due to similar
physiology. There is currently a need for populations of non-human primates with specified
disease susceptible genotypes that cannotbe satisfied by the importation ofanimals from the
wild or by the identification and propagation of valuable founder animals by selective
breeding. In theory, assisted reproductive technologies can be efficiently applied to the rapid
propagation of select animals to satisfy the needs of the biomedical research community.
Ovulation induction protocols utilizing recombinant gonadotropins that produce approxi-
mately 20 fertilizable oocytes per stimulation cycle have been developed for non-human
primates [1,2].Intracytoplasmic sperminjectionisasuccessfulfertilizationprocedure inthis
species [3,4] utilizing one spermatozoa per oocyte and thereby allowing the production of a
very large number of embryos from a single ejaculate. The rapid, efficient propagation of a
valuable founder male, then, might ideally involve cryopreservation of spermatozoa in small
aliquots that retain fertilizing capability similar to that of freshly harvested spermatozoa.
Despite advances in cryopreservation techniques, decreases in progressive motility and
in the percentage of motile spermatozoa are observable after thaw in most species [5,6]
which may be reflected in decreased mucus penetration , decreased in vitro penetration
of salt stored oocytes  and decreased numbers of decondensed spermatozoa heads in
hamster oocytes after IVF or ICSI [9,10]. Most importantly, spermatozoa cryopreservation
results in reduced function, such that conventional insemination is no longer efficient.
damage observed after thaw [6,11]. Internal post-thaw changes in spermatozoa have also
been noted, including decreased mitochondrial membrane potentials  and decreased
chromosomal condensation  which may impact motility, fertilization and subsequent
embryonic development. Reports are inconsistent as to the effectiveness of fresh versus
frozen–thawed spermatozoa in fertilizing oocytes with ICSI. A detailed analysis of clinical
cases utilizing testicular spermatozoa observed that ICSI with fresh, fully developed
spermatozoa had a significantly higher fertilization rate than when frozen–thawed
spermatozoa were used . However, reports on this topic need to be carefully examined
relative to the maturity of the spermatozoa being compared. Mature spermatozoa from
fresh ejaculates yield higher fertilization rates with ICSI than immature spermatozoa of
epididymal or testicular origin whether frozen–thawed or fresh [15–17]. This difference
may relate to the effects of freezing and thawing, or to immaturity; as spermatozoa pass
from the testis to the cauda epididymis, increases in chromosome condensation, motility,
and the abilities to bind zona and activate oocytes are observed [18–20].
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx2
Cryopreservation of spermatozoa from several strains of macaques such as the rhesus
[21–23] and cynomolgus [5,24,25], has been reported. However, no studies have
comparatively evaluated various cryopreservation procedures by fertilization in vitro
(assessed using ICSI) to evaluate cryo-damage beyond that related to motility and binding
to the oocyte membranes. The present report compares four freezing–thawing protocols by
an ongoing effort to identify an approach that supports retention of fertility similar to that
of fresh spermatozoa and allows preservation of a large number of samples from a single
2. Materials and methods
2.1. Animals and collection and evaluation of semen
Use of Laboratory Animals and approved by the Institutional Animal Use and Care
Committee of the Oregon National Primate Research Center, Oregon Health and
Sciences University. Nine adult male rhesus monkeys (6–9 kg) were used in a series of
four semen cryopreservation experiments while developing our current procedures. The
restricted number of oocytes available for this study prevented direct comparisons of
contrasting cryopreservation procedures within oocyte cohorts, as did practical
considerations prevent simultaneous fertility comparisons of fresh and cryopreserved
spermatozoa from the same ejaculate. However, the semen samples utilized throughout
were consistently of high quality, based on sperm motion characteristics and therefore
subset of freezing series data to address if reduced fertilization was a characteristic of
individual males. Additionally, one founder adult male with a highly desirable genotype
housed offsite was involved in the extensive in vivo application of the final
cryopreservation technique. Semen was collected by penile electroejaculation 
andallowedto liquifyat 27–32 8C for 10–15 min. The liquidportionwas harvestedfrom
the coagulum into a 15 mL conical centrifuge tube (05-539-12, Fisher Scientific, Tustin,
CA, USA) and washed twice with 5 mL HEPES-buffered TALP medium (modified
Tyrode’s solution with Albumin, Lactate, and Pyruvate medium; ) containing 0.3%
BSA (TH3) at 130–150 ? g for 5 min and the final pellet was resuspended in 0.25 mL
medium to make up the washed spermatozoa pellet utilized for the different freezing
procedures. Unless otherwise noted, chemicals utilized were purchased from Sigma-
Aldrich, St. Louis, MO, USA. Sperm concentration and motility were evaluated
microscopically with a hemocytometer (100 cells) before freezing and after thawing.
Forward progression was rated on a scale of 1–4, with 1 = no forward progression
(twitching)and4 = rapidprogressivemovement.Onlysemenspecimenswithinitialtotal
motility of more than 70% with progression of at least grade 3 (as observed with a
hemocytometer) were utilized in these experiments.
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx3
2.2.1. Series I
In freeze–thaw series I, the washed sperm pellet was extended with medium consisting
of 6% glycerol and 0.1 M sucrose in TH3 . After gently mixing, the sperm suspension
(in a 15 mL tube) was placed in a 500 mL beaker of 23 8C water and equilibrated to 4 8C in
a refrigerator for 90 min. The suspension was then loaded into 0.25 mL straws on ice
(PETS, Canton, TX, USA) that were sealed by fluid contact with the wick and powder at
one end and heat-crimped at the other end. Straws were then placed horizontally on two
aluminum supports at 1 cm above the LN2(which approximates ?170 8C). The cooling of
liquidwithinastrawto?150 8Coccurredwithin1 minandreached?170 8Cwithin2 min,
as measured by a thermocouple (Tegam, Geneva, OH, USA) placed inside an identical
straw filled with freezing media. Specimens were stored at least 48 h before thawing.
Straws were thawed in ambient air (22 8C) for 15 s before the contents were expelled into
5 mL of TH3 rinse media that approximated a +850 8C/min warming rate. After an
additional wash with 5 mL TH3, count and motility were evaluated.
2.2.2. Series II
Freeze–thaw series II involved diluting the washed pellet to 1 mL with extender
medium consisting of 30% egg yolk, 20% skim milk, 0.06 M glucose in a Tes–Tris buffer
adjusted to a pH 7.4 [5; Dr. C. VandeVoort, University of California, Davis, personal
communication]. A separate portion of the extender medium was then modified to make a
6% glycerated solution of which an equal volume was added to the spermatozoa in thirds
over 30 min at 4 8C, to give a final glycerol concentration of 3%. After equilibration with
the cryoprotectant for a further 60 min at 4 8C, straws were loaded, sealed, frozen in LN2
vapors and stored as with series I. Thawing of straws frozen in experimental series II was
similar to that of series I.
2.2.3. Series III
additional post-thaw purification step. The latter involved density gradient centrifugation
througha columnof50,70and95% Percoll(Amersham Biosciences,Piscataway,NJ,USA)
 before recovery of the 95% fraction and washing twice in 5 mL of TH3.
Additionally, specimens prepared with this procedure were evaluated for within-
ejaculate variability and cryostability of frozen spermatozoa by motility measurements of
spermatozoa from straws held in LN for up to 3 month. Post-thaw retention of motility was
also examined 6 h after thaw processing.
2.2.4. Series IV
Series IVinvolved preparation and equilibration of washed spermatozoa in 3% glycerol
solidified carbon dioxide (dry ice, ?78 8C) forming pellets  with approximate cooling
rates of ?85 and ?60 8C/min for 50 and 100 mL pellets, respectively. After 10 min,
multiple pellets were then placed in dry-ice-cooled cryovials (#2028, Corning, Corning,
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx4
the LN2within a cryovial, placed in a dry test tube suspended in a 37 8C water bath for 40 s
(which approximated a warming rate of 350 8C/min) and then washed in 5 mL of TH3.
Additionally, the effect of pellet size on post-thaw motility was analyzed by comparing
results from five drops frozen at 100, 50 and 20 mL each.
2.3. Post-thaw processing
With all for freeze–thaw approaches, the final washed pellet was suspended in 200 mL
TH3 and post thaw motilities were determined. This suspension was then diluted, if
needed, to 1–3 million motile spermatozoa/mL for ICSI, as previously described [2,4].
Note that in the ICSI experiments comparing fresh versus F/T spermatozoa, for practical
purposes, the same ejaculate could not be used. However, when possible fresh and thawed
spermatozoa from the same male were compared and these cases were additionally
analyzed separately below.
sperm from a genetically valuable male frozen with the series IV protocol. The time from
thawing of the spermatozoa to initiation of ICSI was monitored and evaluated relative to
fertilization rate. The interval from first observation of mature metaphase II oocytes to ICSI
was also recorded, since some oocytes did not mature until several hours after retrieval.
2.4. Follicular stimulation and oocyte collection
Cumulus–oocyte-complexes (COC) were retrieved by follicular aspiration from cycling
females following ovarian stimulation [1,2]. Starting 1–4 days after menses, cycling females
received twice-daily injections of recombinant human FSH (rhFSH; 30 IU i.m., Ares
Advanced Technology, Norwell, MA, USA) and once daily injections of Antide (a GnRH
2 day of rhFSH/Antide stimulation, animals also received twice daily injections of
recombinant human LH (rhLH; 30 IU i.m., Ares Advanced Technology). On the last day of
hormonal stimulation, ovarian morphology was evaluated by ultrasonography (ATL HDI
or greater received an injection of recombinant human chorionic gonadotropin (rhCG; 1000
by mechanical trituration after 1 min exposure to hyaluronidase (1 mg/mL) and held in
CMRL medium (Connaught Medical Research Laboratories media; Life Technologies,
10 mM L-glutamine, 5 mM sodium pyruvate, 1 mM sodium lactate, 100 units/mL of
penicillin,and100 ug/mLstreptomycinforshort-termcultureat37 8Cin5%CO2forupto4 h
prior to ICSI, depending on their maturation status.
2.5. ICSI and embryo culture
The limited number of oocytes available for study necessitated evaluating spermatozoa
from only one freezing procedure on each day of oocyte retrieval. Sibling oocytes were
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx5
always inseminated in parallel with fresh spermatozoa to control for oocyte quality. ICSI
Olympus, Melville, NY, USA) equipped with Hoffman optics and micromanipulators
(Narishige, Tokyo, Japan) housed in a heated room (27–30 8C) with oocytes returned to
culture within 10 min. The micromanipulation chamber (Falcon 1009, Becton-Dickinson,
Franklin Lakes, NJ, USA) contained oil and two drops: a sperm drop consisting of 4 mL of
10% polyvinylpyrrolidone (Irvine Scientific, Santa Ana, CA, USA) in TALP/HEPES and
1 mL of spermatozoa (3 million/mL) and an oocytedrop, 20 mLof TH3, intowhich mature
oocytes were placed. Individual spermatozoa were immobilized by striking the tail and
then injected into oocytes away from the polar body using a 7 mm outer diameter
micropipette (Humagen, Charlettesville, VA, USA). Only progressively motile sperma-
a monolayer of Buffalo Rat Liver cells (BRL; American Type Culture Collection,
Manassas, VA, USA) (2.5 ? 104cells per well) in 4-well dishes (Nalge Nunc International
Co, Naperville, IL, USA) containing 0.6 mL of CMRL culture medium with 10% FBS at
37 8C in 5% CO2. Embryos were transferred to fresh plates of BRL cells every other day.
Pronuclear formation was recorded 10–16 h post-ICSI and the progression of embryo
growth was recorded daily.Fertilization was defined as pronuclear formation and/or timely
cleavagewith nucleated blastomeres. Blastocyst formation was defined as the expansion of
the compacted embryo with cavitation to include both a discernible trophectoderm and an
inner cell mass.
2.6. Embryo transfer
Embryo transfers were conducted on adult, multiparous rhesus females in which blood
samples were collected daily for measurement of estradiol beginning 8 day after detection
ofmenses.Estradiollevelswere measured withanautomatedimmunoassay(Elecsys 2010,
Roche Diagnostics, Laval, Que., Canada) in order to detect the mid-cycle estradiol peak.
Two to five days after the peak, animals were prepared for transcervical embryo transfers.
Under ketamine anesthesia (10 mg/kg, i.m.; Fort Dodge Laboratories, Fort Dodge, IA,
USA) animals were positioned in sternal recumbency, and a polyvinyl catheter aided by a
stylet(Pattonmodel, Cook OB/GYN,Spencer,IN,USA)was gentlyguidedintothe uterus.
Alternative tubal transfers were via surgical or laparoscopic approaches under isoflurane
gas anesthesia (Henry Schein, Sparks, NV, USA) while monitoring heart rate and
peripheral oxygen saturation. Two embryos in 15 mL of TH3 medium were then slowly
ejected. Pregnancy detection involved monitoring weekly hormone levels for three weeks.
Ultrasonography (Philips Medical Systems) was initially performed on day 25 to detect a
fetal heartbeat. Fetal status was further monitored at trimester intervals by ultrasound for
the duration of pregnancy.
2.7. Data analysis
Sperm motility proportions were transformed to the arcsine of the square root before
comparing byANOVAandFisher’sprotected leastsignificant differencetestwithStatview
software (SAS Institute Inc., Cary, NC, USA). Sperm motilty grades were converted to
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx6
proportions of the range and similarly transformed before ANOVA analysis. Fertilization
rates and embryo development rates within treatment series were also transformed to the
arcsine of the square root of proportions, prior to comparison between fresh and frozen
results using the Student’s t-test (SAS). The regression analysis offertilization rates versus
incubation time post-thawwas evaluated using ANOVA (SAS). Values with P < 0.05 were
considered different in all cases.
3.1. Motility of fresh specimens
In series I–IV, the 26 fresh semen specimens from nine males collected for these
experiments had similar motilities after washing with the percent motile and forward
progression ranging from 77 to 81% and 3 to 3.5, respectively. These data were combined
for presentation and provided control values for comparison to post-thaw measurements of
specimens after different cryopreservation procedures (Fig. 1).
3.2. Series I
After thawing the eight specimens frozen in series I, 17 ? 6% of the spermatozoa were
motile and the forward progression (1.4 ? 0.1) was significantly lower than non-frozen
control samples (Fig. 1). In four ICSI trials which evaluated the fertility potential of
frozen–thawed spermatozoa from three males prepared in series I, reduced fertility in
sibling oocytes was observed when compared to control, non-frozen spermatozoa (40%,
n = 40 versus 77%, n = 39; P < 0.05; Table 1). The blastocyst developmental rate after 8
daysofculturefromtheembryosfertilizedwiththawedspermatozoawas56%(n = 16)and
with fresh spermatozoa, 57% (n = 30).
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx7
Fig. 1. Recovery of rhesus monkey spermatozoa motility and progression after cryopreservation. Data is
presented as means ? S.E. of 8 frozen specimens per frozen–thawed treatment and 26 fresh controls. Different
letters or symbols within the measured parameter indicate a difference among treatments (P < 0.05).
3.3. Series II
Eight specimens from four males cryopreserved with the procedure in experimental
series II had similar motility (24 ? 5%), but higher forward progression (2.2 ? 0.1,
P < 0.05; Fig. 1). However, four ICSI trials in series II using thawed spermatozoa, also
yielded a reduced fertilization rate (44%, n = 32) when compared to sibling oocytes
injected with control spermatozoa (86%, n = 35; P < 0.05) (Table 1). The blastocyst
development rate from embryos fertilized with thawed spermatozoa was 57% (n = 7)
compared to 50% (n = 30) with fresh spermatozoa.
3.4. Series III
thawingofeight ejaculatesfromthree males,similartothatinseriesII,butfurther processed
by Percoll purification. This processing resulted in motility (75 ? 3%, P < 0.01) and
progression scores (2.8 + 0.1, P < 0.05) similar to those of control samples (Fig. 1). When
these elite frozen–thawed spermatozoa were used in six ICSI experiments, the fertilization
rate was 49% (n = 61), still less than that with sibling oocytes injected with freshly prepared
spermatozoa, 77% (n = 52, P < 0.01; Table 1). The blastocyst development rate was 54%
(n = 24) with frozen–thawed spermatozoa and 50% (n = 30) with fresh spermatozoa.
Using the procedure from series III, the within-ejaculate variability and cryostability of
frozen spermatozoa from three males was evaluated by motility measurements of
spermatozoa from nine straws held in LN for up to 3 month. The thawed motility
percentages had a coefficient of variation of 4%, suggesting minimal degradation.
Although ICSI was generally performed within 2 h of sperm recovery, it was also of
processed with the procedure from series III, high motility (81 ? 3%; 2.5 progression) was
maintained through 2 hof culture;after 6 h, 72 ? 12% of the cells werestill motile, though
of low forward progression.
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx8
IV compared to ICSI with fresh spermatozoa
I (4 trials)Frozen–thawed
16/40 (40%) a
30/39 (77%) b
II (4 trials)Frozen–thawed
14/32 (44%) a
30/35 (86%) b
III (6 trials)Frozen–thawed
30/61 (49%) a
40/52 (77%) b
IV (6 trials)Frozen–thawed
47/65 (73%) b
45/53 (84%) b
Different letters within columns indicate a difference among values (P < 0.05).
aBlastocystsfromembryos.Inonetrialfrom seriesIIandtwo fromseriesIIIandIV,embryoswereutilizedfor
other studies and unavailable for developmental analysis.
3.5. Series IV
As an alternative to LN-based, low-temperature freezing in straws, cooling of extended
spermatozoa as pellets on dry icewas examined in series IV with nine specimens from four
males. This approach was associated with diminished post-thaw percent motility
(49 ? 5%) versus fresh spermatozoa (P < 0.05), similar to the outcomes from
experimental series I and II. However, forward progression of spermatozoa from thawed
pellets showed forward progression (3.2 ? 0.1) compared to fresh or Percoll isolated
specimens (Fig. 1). The fertility analysis in six ICSI trials using spermatozoa frozen as
pellets in series IV yielded a higher fertilization rate (73%, n = 65 oocytes) compared with
frozen–thawed spermatozoa via other procedures (P < 0.01) which was not significantly
different from levels obtained with control spermatozoa in sibling oocytes (84%, n = 53
oocytes; Table 1). The blastocyst development rate was 32% (n = 44), with embryos from
spermatozoa frozen in series IVand 35% (n = 43) with fresh spermatozoa. All six initial
trials in series IV paired fresh spermatozoa with F/T spermatozoa from the same male.
The effect of pellet size on post-thaw motility was analyzed separately by comparing
results from five drops frozen at 100, 50 and 20 mL each. The post-thaw motility ranged
post-thaw motility within this range of drop sizes.
3.6. Same-male comparison
Twenty-four of the 27 trials conducted with spermatozoa processed in series I, II and III
were matched with spermatozoa from the same male for both fresh and F/T ICSI albeit
different specimens. An analysis of fertilization results for these 24 experiments where the
same male was employed found similarly reduced fertilization when post-thaw motile
spermatozoa were injected (43%, n = 79 oocytes) compared to the rate using control
spermatozoa (82%, n = 88 oocytes; P < 0.05). This observation indicates that reduced
fertilization with frozen–thawed spermatozoa from experimental procedures in series I, II
and III is a characteristic of most if not all the males tested.
3.7. Post-thaw incubation
In a subsequent application of the method derived from series IV, semen was collected
from a genetically valuable male at the New England National Primate Research Center
and spermatozoa were cryopreserved in pellets for transportation to our laboratory. Forty-
one pellets frozen from several collections were thawed with a motility of 39 ? 1% and
when used for ICSI of 542 oocytes, an overall fertilization rate of 50.7 ? 4.4%. This
fertility suggested that this particular male was subpar regarding the tolerance of its
spermatozoa to freezing and thawing relative to our previous results with good quality
spermatozoa. A study was then done evaluating the time from thawing of the spermatozoa
to initiationof ICSI. Therewas a correlation (P < 0.01) betweenincreased incubation time
of thawed spermatozoa prior to ICSI and fertilization (Fig. 2). Incubation times <2 h
(n = 18) resulted in 32.7 ? 4.4% fertilization, in contrast to those thaws used ?3 h (n = 15)
which had 73.9 ? 6.7% fertilization. Oocyte age post-collection did not appear to
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx9
contributetothisoutcome,asevidenced byacomparison betweentheperformancesofMII
oocytes that were matured at collection versus those that matured late, in vitro.
3.8. Embryo transfer
frozen–thawed spermatozoa from series IV protocol involved five transcervical transfers.
Two clinical pregnancies resulted in a healthy male weighing 520 grams and a healthy
female weighing 385 grams delivered at 159 and 158 day of gestation, respectively,
establishing the first primates born using ejaculated frozen–thawed spermatozoa and ICSI
insemination. In the ongoing series utilizing frozen–thawed spermatozoa from the
genetically unique male at the New England National Primate Research Center, 101
embryo transfers of 1–2 early cleavage stage embryos into recipients 2–3 day after
ovulation has resulted in 21 additional pregnancies and live births.
We have developed improved cryostorage procedures for rhesus monkey spermatozoa
to be used for ICSI in assisted reproduction providing numerous thaws of adequate
spermatozoa numbers from a few ejaculates. This is the first study comparing freezing
procedures that not only assessed motility, but also evaluated the fertilizing capability of
thawed ejaculated spermatozoa using ICSI (thereby circumventing motility and zona
binding). We found that motility of spermatozoa frozen in straws in LN vapors did not
show a relationship with fertilization, suggesting the existence of sub-lethal, cryo-damage
with motility characteristics similar to freshly prepared spermatozoa, did not improve ICSI
fertilization rates to levels achieved with fresh spermatozoa. An alternative procedure that
involved relatively slower cooling and warming was associated with the retention of ICSI
fertilizing capability, similar to that of fresh spermatozoa. Additionally, a 3–4 h incubation
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx 10
Fig. 2. Plot of fertilization rates (%) from 41 experiments with respective incubation time post-thaw in hours
before spermatozoa was utilized for ICSI (R, 0.45; P, <0.001).
after thawing improved the fertilization rate with spermatozoa from a male with poor
recovery of motility post-thaw thaw.
Although current cryopreservation procedures are directly lethal to a considerable
proportion of spermatozoa in a semen sample, the breadth of sub-lethal, cryo-damage,
not been as obvious . Evaluations offertility post-thaw of ejaculated spermatozoa have
included both motility and oocyte penetration, but have not examined factors unrelated to
motility, as is possible with ICSI [5,22,23]. During cryopreservation procedures, freezing
rates and cryoprotective agents interact to effect cellular membrane fluidity and water
channel permeability contributing to the level of cell dehydration important for post-thaw
survival [32–35]. Membranes of subcellular structures throughout the cell may be
compromised by the physical state changes, as shown in electron microscopy studies of
frozen–thawed primate spermatozoa . Moreover, reduced fertilization may be related
to loss of soluble, spermatozoa-associated, oocyte activation factors, since isolation
protocols for these factors utilize repeated freezing and thawing of spermatozoa [4,36]. An
aggressive freeze–thaw regimen diminished the oocyte activating ability of human
spermatozoa injected into hamster oocytes; this deficiency was overcome by
supplementation with a sperm cytosolic factor [37,38]. Sperm-borne, oocyte activating
factors have also been associated with perinuclear structures and while these structures are
relatively insoluble [39–41], they still may be compromised during the freezing procedure
. Another possible site of freeze–thaw damage is the disulfide bond of the protamines
that replace histones during DNA compaction in spermatogenesis; sperm freezing and
thawing may reduce chromosomal compaction post-thaw relative to fresh spermatozoa
diminish pronuclear formation or blastocystdevelopment when spermatozoawere injected
into oocytes  suggesting that reduced chromosomal compaction may not prominently
influence fertilization. In the present study, the best fertilization results were obtained from
spermatozoa crypreserved as small drops on dry ice, as compared to spermatozoa frozen in
straws suspended in liquid nitrogen vapors. Inherent in the dry ice procedure is a reduced
cooling rate of ?60 to ?85 8C/min compared to that occurring in straws in LN vapor
(?150 8C/min). A reduced cooling rate has also been reported to be optimal in studies with
mice spermatozoa which incur a dramatic fall in motility above ?100 8C/min .
A reduced ability of the spermatozoa to activate oocytes is considered the main cause of
ICSI-related fertilization failures, including incomplete chromosome decondensation and
premature condensation [44–47]. Oocyte activation mechanisms involve calcium
oscillations, which inactivate the maturation promoting factor, trigger sperm head
decondensation and, subsequently, promote pronuclear membrane formation .
Fertilization by ICSI bypasses the interaction of sperm and oocyte membranes, however,
the micropipette penetration of the oolemma and aspiration of cytoplasm into the
micropipette is thought to simulate the initial sensitization to sperm factors [48,49]. Our
present ICSI procedures with fresh spermatozoa resulted in fertilization success as good as
or better than can we have previously achieved with invitro fertilization (50–75%) . In
vitro fertilization in the rhesus monkey is hampered by the numerous pre-activation steps
required to capacitate spermatozoa and a propensity of the spermatozoa to agglutinate
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx11
The significant improvement in ICSI fertilization rates with short-term culture for a few
hours post-thaw observed with spermatozoa from a genetically valuable male, is
noteworthy. Although natural mating was successful with this male, his spermatozoa were
exceptionally susceptible to cryodamage (similar to other individuals of other species).
Cryopreservation is known to effect sperm membrane lipids, ion channels and enzymes
[53–55]. Since oocyte activating factors are sequestered in the postacrosomal perinuclear
theca [40,56], they also are likely perturbed during the freeze–thaw cycle, perhaps more so
in this individual. Enzyme activity such as phosphorylation of acrosomal components is
known to occur during capacitation [57,58] and possibly this brief in vitro incubation
allowed adequate recovery of necessary mechanisms in these markedly cryo-sensitive
spermatozoa. Our incubationof thawed spermatozoa fora fewhours after freezing indrops
on dry ice has resulted in 21 additional live births from this genetically unique male .
In contrast to the range of fertilization rates observed after ICSI with frozen–thawed
spermatozoa in the present experiments, in vitro embryonic development was similar
between embryos produced using fresh or frozen–thawed spermatozoa. These blastocyst
developmental rates were also similar to those obtained previously in the rhesus monkey
with fresh spermatozoa by this and other laboratories, whether inseminations were by
either ICSI or IVF [2,4,60,61]. Additionally, our successful production of 23 pregnancies
from 106 embryo transfers confirms functional viability of frozen–thawed monkey sperm
and is similar to the pregnancy rates we reported using embryos produced with fresh
spermatozoa [2,62]. Reports in the human utilizing frozen–thawed spermatozoa have also
noted no difference in embryonic development with implantation rates compared to those
achieved for fresh spermatozoa [15,17,63]. The present results augmented by the above
literature suggests that once fertilization is established in the primate, post fertilization
embryonic development is minimally impacted by prior cryopreservation of paternal DNA
with these methods. However, as shown in the mouse, suboptimal freezing procedures or,
perhaps, freezing of sensitive spermatozoa from specific individuals  may affect
subsequent fetal development, a possibility that was not intensively investigated here.
Freezing in pellets has an additional advantage relative to straws; it allows reduced
volumes to be stored and retrieved separately. The retention of fertilization by ICSI with
spermatozoa cryopreserved in small pellets provides the ability to extend the number of
specimens 10 times that employed with straws. This advantage is important when there is
limited availability of spermatozoa from a male with highly desirable genetic traits.
Additionally, the use of the same ejaculate for several fertilization experiments would
produce less variability from the male contribution. The room air freezing of pellets on dry
ice was a concern due to the potential for contamination. However, no problems were
encountered. The use of sterile dry ice and spermatozoa processing in a sterile hood could
of course be considered.
In summary, the present experiments in the rhesus monkey utilized ICSI as a tool to
detect sub-lethal cryo-damage and found reduced fertilization unrelated to post-thaw
motility with spermatozoa frozen in straws in LN vapors. An alternative procedure,
freezing specimens in drops on dry ice preserved fertility similar to that of fresh
spermatozoa. The latter approach also offers the advantage of a marked increase in the
number of specimens that can be obtained per ejaculate. Sperm cryopreservation, when
combined with ICSI, will enable a multitude of embryos to be produced from a single
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx12
ejaculate. Proof of principle for this approach in the rhesus monkey was provided by 23
pregnancies to date. The creation of viable embryos by applying the ARTs with
cryopreserved spermatozoa should allow the propagation of nonhuman primate disease
models that are in great demand by the biomedical research community.
The authors gratefully acknowledge the contributions of Dr. David Hess for hormone
assays and Dr. John Fanton and the Division of Animal Resources for surgical procedures
and animal care. The Assisted Reproduction core is recognized for assistance in providing
semen samples. The able technical assistance of Andrea Widmann-Browning and Cathy
Ramsey is deeply appreciated. The open discussions of freezing protocols with Dr. Cathy
A. VandeVoort, University of California, Davis, were of value as were comments on the
manuscript by Dr. Richard Stouffer. Supported by NIH RR12804 and NIH RR 16030 to
D.P. Wolf, A142709 and Ares Advanced Technology, Inc., Norwell, MA.
 Zelinski-Wooten MB, Hutchison JS, Hess DL, Wolf DP, Stouffer RL. Follicle stimulating hormone alone
supports follicle growth and oocyte development in gonadotrophin-releasing hormone antagonist-treated
monkeys. Hum Reprod 1995;10:1658–66.
 Nusser KD, Mitalipov S, Widmann A, Gerami-Naini B, Yeoman RR, Wolf DP. Developmental competence
of oocytes after ICSI in the rhesus monkey. Hum Reprod 2001;16:130–7.
 Hewitson L, Takahashi D, Dominko R, Simerly C, Schatten G. Fertilization and embryo development to
blastocysts after intracytoplasmic spermatozoa injection in the rhesus monkey. Hum Reprod 1998;13:3449–55.
 Meng L, Wolf D. Spermatozoa-induced oocyte activation in the rhesus monkey: nuclear and cytoplasmic
changes following intracytoplasmic spermatozoa injection. Hum Reprod 1997;12:1062–8.
 Tollner TL, VandeVoort CA, Overstreet JW, Drobnis EZ. Cryopreservation of spermatozoaatozoa from
cynomolgus monkeys (Macaca fascicularis). J Reprod Fertil 1990;90:347–52.
 McLaughlin EA, Ford WC, HuLl MG. Motility characteristics and membrane integrity of cryopreserved
human spermatozoaatozoa. J Reprod Fertil 1992;92:527–34.
 Urry RL, Carrel DT, Hull DB, Middleton RG, Wiltbank MC. Penetration of zona-free hamster ova and
bovine cervical mucus by fresh and frozen human spermatozoaatozoa. Fertil Steril 1983;39:690–4.
 Sankai T, Terao K, Yanagimachi R, Cho F, Yoshikawa Y. Cryopreservation of spermatozoaatozoa from
cynomolgus monkeys (Macaca fascicularis). J Reprod Fertil 1994;101:273–8.
 Johnson A, Hessel P. The potential for cryopreserved spermatozoa to undergo capacitation as determined by
the optimized spermatozoa penetration assay. Fertil Steril 1995;64(Suppl.):168.
 Aslam I, Fishel S. Evaluation of the fertilization potential of freshly isolated, in-vitro cultured and
cryopreserved human spermatids by injection into hamster oocytes. Hum Reprod 1999;14:1528–33.
 Ellington JE, Broemeling LD, Broder SJ, Jones AE, Choker DA, Wright RW. Comparison of fresh and
cryopreserved human spermatozoa attachment to bovine oviduct (uterine tube) epithelial cells in vitro. J
 Thomas CA, Garner DL, DeJarnette JM, Marshall CE. Effect of cryopreservation of bovine spermatozoa
organelle function and viability as determined by flow cytometry. Biol Reprod 1998;38:786–93.
 Hammadeh ME, Askari AS, Georg T, Rosenbaum P, Schmidt W. Effect of freeze-thawing procedure on
chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and
subfertile men. Int J Androl 1999;22:155–62.
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx 13
 Sousa M, Cremades N, Silva J, Oliveira C, Ferraz L, Teixeira da Silva J, et al. Predictive value of testicular
histologyin secretoryazoospermic subgroups and clinical outcome after microinjection offreshand frozen–
thawed sperm and spermatids. Hum Reprod 2002;17:1800–10.
 Nagy Z, Liu J, Cecile J, Silber S, Devroey P, Van Steirteghem A. Using ejaculated, fresh, and frozen–thawed
epididymal and testicular spermatozoaatozoa gives rise to comparable results after intracytoplasmic
spermatozoa injection. Fertil Steril 1995;63:808–15.
 Van Steirteghem A, Nagy P, Joris H, Janssenswillen C, Staessen C, Verheyen G, et al. Results of
intracytoplasmic spermatozoa injection with ejaculated, fresh, and frozen–thawed epididymal and testicular
spermatozoaatozoa. Hum Reprod 1998;13(Suppl.):134–42.
 Habermann H, Seo R, Cieslak J, Niederberger C, Prins GS, Ross L. In vitro fertilization outcomes after
intracytoplasmic spermatozoa injection with fresh or frozen–thawed testicular spermatozoaatozoa. Fertil
 Yeung CH, Morrell JM, Cooper TG, Weinbauer GF, Hodges JK, Nieschlag E. Maturation of spermatozoa
motility in the epididymis of the common marmoset (Callithriz jacchus) and the cynomolgus monkey
(Macacca fascucularis). Int J Androl 1996;19:113–21.
 Golan R, Cooper TG, Oschry Y, Oberpenning F, Schulze H, Shochat L, et al. Changes in chromatin
condensation of human spermatozoa during epididymal transit as determined by flow cytometry. Hum
 Hammadeh ME, al-Hasani S, Doerr S, Stieber M, Rosenbaum P, Schmidt W, et al. Comparison between
chromatin condensation and morphology from testis biopsy extracted and ejaculated spermatozoa and their
relationship to ICSI outcome. Hum Reprod 1999;14:363–7.
 Leverage WE, Valerio DA, SchuLtz AP, Kingsbury E, Dorey C. Comparative study on the freeze
preservation of spermatozoa. Primate, bovine, and human. Lab Anim Sci 1972;22:882–9.
 Si W, Zheng P, Tang X, He X, Wang H, Bavister BD, et al. Cryopreservation of rhesus macaque (Macaca
mulatta) spermatozoaatozoa and their functional assessment by in vitro fertilization. Cryobiology
 Sanchez-Partida GL, Maginnis G, Domminko T, Martinovich C, McVay B, Fanton J, et al. Live rhesus
offspring by artificial insemination using fresh spermatozoa and cryopreserved spermatozoa. Biol Reprod
 Feradis AH, Pawitri D, Suatha IK, Amin MR, Yusuf RL, Sajuthi D, et al. Cryopreservation of epididymal
spermatozoaatozoa collected by needle biopsy from cynomolgus monkeys (Macaca fascicularis). J Med
 Ng SC, Martelli P, Liow SL, Herbert S, Oh SH. Intracytoplasmic injection of frozen–thawed epididymal
spermatozoa in a nonhuman primate mode, the cynomolgusmonkey (Macaca fascicularis). Theriogenology
 Lanzendorf SE, Gliessman PM, Archibong AE, Alexander M, Wolf DP. Collection and quality of rhesus
monkey semen. Mol Reprod Dev 1990;25:61–6.
 Boatman DE. In vitro growth of non-human primate pre- and peri-implantation embryos. In: Bavister BD,
Plenum Press; 1987. p. 273–308.
 Larson JM, McKinney KA, Mixon BA, Burry KA, Wolf DP. An intrauterine insemination-ready cryopre-
servation method compared with spermatozoa recovery after conventional freezing and post-thaw proces-
sing. Fertil Steril 1997;68:143–8.
 McClure RD, Nunes SL, Tom R. Semen manipulation: improved spermatozoa recovery and function with a
two-layer Percoll gradient. Fertil Steril 1989;51:874–9.
 Holt WV. Fundamental aspects of spermatozoa cryobiology: the importance of species and individual
differences. Theriogenology 2000;53:47–58.
 Henry MA, Noiles DD, Gao D, Mazur P, Critser JK. Cryopreservation of human spermatozoaatozoa IV. The
effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity, and
mitochondrial function. Fertil Steril 1993;60:911–8.
 Giraud MN, Motta C, Boucher D, GrizardG. Membranefluidity predictsthe outcome of cryopreservation of
human spermatozoa. Hum Reprod 2000;15:2160–4.
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx14
 Gilmore JA, Liu J, Woods EJ, Peter AT, Critser JK. Cryoprotective agent and temperature effects on human Download full-text
spermatozoa membrane permeabilities: convergence of theoretical and empirical approaches for optimal
cryopreservation methods. Human Reprod 2000;15:334–43.
 IshibashiK,KuwaharaM, GuY,KageyamaY,TohsakaA,SuzukiF, etal.Cloningand functional expression
 Swann K. A cytosolic spermatozoa factor stimulates repetitive calcium increases and mimics fertilization in
hamster eggs. Development 1990;110:1295–302.
 Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool 1999;284:696–704.
 Ahmadi A, Ng SC. Developmental capacity of damaged spermatozoa. Hum Reprod 1999;14:2279–85.
 Oko R. Developmental expression and possible role of perinuclear theca proteins in mammalian sperma-
tozoa. Reprod Fertil Dev 1995;7:777–97.
activation suggests the invovlement of spermatozoa perinuclear material. Biol Reprod 1998;58:1407–15.
 Perry ACE, Wakayama T, Yanagimachi R. A novel trans-complementation assay suggests full mammalian
oocyte activation is coordinately initiated by multiple,submembranespermatozoa components. Biol Reprod
 Oko R, AuL RB, Sutovsky P. The spermatozoa head cytoskeleton. In: Robaire B, Chemes H, Morales CR.,
editors. Andrologyin the 21st Century, Proceedings of the VIIth International Congress of Andrology. 2001.
 KoshimotoC,MazurP.Effectsofcoolingandwarmingratetoandfrom?70 8C,andeffectoffurthercooling
from ?70 to ?196 8C on the motility of mouse spermatozoa. Biol Reprod 2002;66:1477–84.
 Sousa M, Tesarik J. Ultrastructural analysis of fertilization failure after intracytoplasmic spermatozoa
injection. Hum Reprod 1994;9:2374–80.
 Hewitson LC, Simerly CR, Tengowski MW, Sutovsky P, Navara CS, Haavisto AJ, et al. Microtubule and
chromatin configurations during rhesus intracytoplasmic spermatozoa injection: successes and failures. Biol
 Rawe VY, Olmedo SB, Nodar FN, Doncel GD, Acosta AA, VituLlo AD. Cytoskeletal organization defects
and abortive activation in human oocytes after IVF and ICSI failure. Mol Hum Reprod 2000;6:510–6.
 Ozil J-P. Role of calcium oscillations in mammalian egg activation: experimental approach. Biophys Chem
 Palermo G, Joris H, Derde MP, Camus M, Devroey P, Van Steirteghem A. Spermatozoa characteristics and
outcome of human assisted fertilization by subzonal insemination and intracytoplasmic spermatozoa
injection. Fertil Steril 1993;59:826–35.
 Dozortsev D, Rybouchkin A, De Sutter P, Dhont M. Spermatozoa plasma membrane damage prior to
intracytoplasmic spermatozoa injection: a necessary condition for spermatozoa nucleus decondensation.
Hum Reprod 1995;10:2960–4.
 Wolf DP, Vandervoort CA, Meyer-Haas GR, Zelinski-Wooten MB, Hess DL, Baughman WL, et al. In vitro
fertilization and embryo transfer in the rhesus monkey. Biol Reprod 1989;41:335–46.
 Bavister BD, Boatman DE, Leibfried ML, Loose M, Vernon MW. Fertilization and cleavage of rhesus
monkey oocytes in vitro. Biol Reprod 1983;28:983–99.
 Boatman DE, Bavister BD. Stimulation of rhesus monkey spermatozoa capacitation by cyclic nucleotide
mediators. J Reprod Fertil 1984;71:357–66.
 Glander HJ, Schaller J. Hidden effects of cryopreservation on quality of human spermatozoa. Cell Tiss Bank
 De La Cueva FI, Rigau R, Pujol R, Peidrafita J, Rodriguez-Gil JE. Resistance to hyperosmotic stress in boar
spermatozoaatozoa: the role of the ionic pumps and the relationship with cryosurvival. Anim Reprod Sci
 RossatoM,ZorziM, FerlinA,Garolla A,Foresta C. Effectsofcryopreservationonprogesterone-inducedion
fluxes and acrosome reaction in human spermatozoa. Hum Reprod 2000;15:1739–43.
 Manandhar G, Toshimori K. Fate of postacrosomal perinuclear theca recognized by monoclonal antibody
MN13 after sperm head microinjection and its role in oocyte activation in mice. Biol Reprod 2003;68:655–
R.R. Yeoman et al./Theriogenology xxx (2004) xxx–xxx15