1. Introduction
Freezing mammalian spermatozoa offers many advantages to the livestock industry, particularly in conjunction with genetic evaluation and selection programmes, such as in
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sire reference schemes Maxwell, 1984 . However, the biggest obstacle to the exploita- tion of frozen semen is that freezing and thawing of spermatozoa of any species
generally leads to a decrease in the percentage of motile cells post-thawing as a result of Ž
. damage to membrane structures Quinn et al., 1969; Nath, 1972 . As a consequence,
fertility following artificial insemination is poorer than with fresh semen in most species and can only be partially compensated for by using greater numbers of spermatozoa in
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the insemination dose Watson, 1995 . Damage to sperm membranes primarily occurs during the freezing and thawing
process over the temperature range y158C to y608C and not during storage in liquid Ž
. nitrogen Mazur, 1965 . In the case of ram spermatozoa, most damage occurs between
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y108C and y258C; the region of ice crystallisation Salamon and Maxwell, 1995 . The process of cell dehydration that accompanies slow freezing is potentially beneficial for
cell survival, whereas rapid freezing rates are considered more likely to cause cell death Ž
. Watson, 1995 .
There are some reports in the literature dealing with the effects of freezing rate on Ž
post-thaw motility and acrosome integrity Watson and Martin, 1975; Fiser and Fairfull, .
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Ž 1986 . O’Neill 1998 observed that semen frozen rapidly from 58C to y258C at
. y58Crmin
had significantly better viability, mitochondrial activity and acrosome Ž
. integrity than after a slower y0.58Crmin freezing rate over the same interval. In these
studies, there was no direct evaluation of the fertilising ability of the sperm. Therefore, the objective of this study was to re-examine the effects of freezing rate, described by
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O’Neill 1998 , in the context of testing in vitro procedures for evaluating the fertilising capacity of frozen-thawed ram spermatozoa. The overall aim was to provide a method
for evaluating fertilizing ability of ram spermatozoa without the need to resort to in vivo methods. The procedure employed the techniques of in vitro maturation, fertilisation and
culture to assess the fertilisation rate and developmental competence of ovine oocytes. The procedure was validated by parallel in vivo studies using adult ewes.
2. Materials and methods
2.1. Semen preparation and freezing Semen was collected, by artificial vagina, from six mature rams known to have good
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fertility. Following collection, motility wave motion was examined and graded on a Ž
scale of 0–5 0 s non-motile, 5 s dense semen with highly vigorous motility; Smith et .
al., 1979 . Any sample with a wave motion below 3 was discarded. The concentration of Ž
spermatozoa in the sample was assessed using a previously calibrated colorimeter EEL, .
Halstead, Essex . Semen was diluted up to 7.5 ml with an extender, based on skim milk
and egg yolk, which was maintained at 358C. The samples were then placed in a cold Ž
. room 58C and allowed to cool slowly to 58C. A second extender, containing 14
glycerol, was then added in two-step fashion and left to equilibrate for 90 min. The Ž
. Ž
. diluted samples were placed in a refrigerated 58C centrifuge Sorvall RC-3B Plus and
centrifuged for 15 min at 700 = g. Following centrifugation, the supernatant was removed and the pellet was resuspended in the freezing extender, containing 7
glycerol, to a volume that would contain 800 = 10
6
spermatozoarml. The semen was Ž
. then loaded into 0.25-ml straws Minitub, Oberkochen, Germany . The straws from each
ejaculate were randomly and equally allocated to one of the two freezing regimes: ‘‘slow’’-frozen semen was cooled from 58C to y258C at y0.58Crmin, while ‘‘fast’’-
frozen semen was cooled at y58Crmin over the same temperature range. Cooling from y258C to y1308C was at a rate of y508Crmin for both treatments. A programmable
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freezer Planar Series II that had been pre-cooled to 58C was used. Once the straws reached y1308C, they were plunged directly into liquid nitrogen for storage.
2.2. Experiment 1: in Õitro fertilisation IVF 2.2.1. Oocyte maturation in Õitro
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Chemicals were obtained from Sigma St. Louis, MO, USA
unless otherwise indicated. The conditions for maturation were a slightly modified version of those
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described by Lonergan et al. 1996 . Briefly, a stock solution of epidermal growth factor Ž
. Ž
. Ž
. EGF was prepared 10 mgrml in medium 199 M199 and stored at y208C until use,
at which time it was diluted to a working concentration of 10 ngrml. Ovaries were Ž
. collected from slaughtered ewes during the breeding season and placed in phosphate-
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buffered saline PBS at approximately 308C for transport to the laboratory. After Ž
. washing in fresh PBS, cumulus oocyte complexes
COCs were recovered from
surface-visible follicles by aspiration into sterile tubes containing 2 ml of aspiration medium. After all ovaries had been aspirated, COCs were located under a stereomicro-
scope and passed through two washes of aspiration medium followed by three washes in Ž
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modified PBS supplemented with pyruvate 36 mgrml , gentamycin 50 mgrml and Ž
. bovine serum albumin 0.5 mgrml; Sigma, fraction V, cat. a A-9647 . Groups of COCs
Ž were then transferred into four-well plates
50 COCs per well, Nunc, Roskilde, .
Ž Denmark containing 500 ml of maturation medium M199 q 10 ngrml EGF q 10
. fetal calf serum and incubated for 24 h in 5 CO in humidified air at 398C.
2
2.2.2. In Õitro fertilisation For IVF, matured COCs were washed four times in PBS and twice in synthetic
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oviduct fluid medium SOFB; O’Brien et al., 1997 . Oocytes were then transferred into Ž
. four-well plates containing 250 ml of SOFB medium per well 50 oocytes per well . For
sperm preparation, three straws, representing one ram and a particular treatment, were Ž
. thawed 708C for 8 s and pooled. A second pool was formed using straws from the
same ram but representing the other freezing treatment. Motile spermatozoa were Ž
. obtained by centrifugation, at 700 = g, on a Percoll Pharmacia, Uppsala, Sweden
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discontinuous density gradient 2 ml of 45 Percoll over 2 ml of 90 Percoll for 20
min at room temperature. Spermatozoa collected at the bottom of the 90 fraction were washed in SOFB medium and pelleted by centrifugation at 100 = g for 10 min at room
temperature. The spermatozoa were counted on a haemocytometer and diluted in the appropriate volume of SOFB to yield a concentration of 2 = 10
6
spermatozoarml. A 250-ml aliquot of this suspension was added to each well, giving a final concentration of
1 = 10
6
spermatozoarml. The plates were then incubated for 20–24 h, in 5 CO in
2
humidified air, at 398C. The COCs obtained form a given batch of ovaries constituted a replicate; spermatozoa from two rams at most were used in each replicate. Spermatozoa
from each ram were represented in at least two replicates. A total of nine replicates was involved in this experiment.
2.2.3. In Õitro culture Embryo culture was carried out in modified SOF medium under paraffin oil in a
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humidified atmosphere of 5 CO , 5 O and 90 N at 398C Carolan et al., 1995 .
2 2
2
Twenty-four hours post insemination, presumptive zygotes were denuded by repeated pipetting, and washed four times in PBS and in SOF before being transferred, in groups
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of 20–30, into culture droplets one zygote per microliter of medium . Fetal calf serum Ž
. Ž
FCS; 10 vrv was added to each droplet 24 h after placement in culture i.e. 48 h .
post insemination . Cleavage rate was assessed 48 h after placement in culture. The number of embryos
that developed to the blastocyst stage was assessed on days 6 and 8 post insemination. On day, 8 the embryos were placed on slides, air-dried and fixed overnight in ethanol
Ž Ž
. and then stained using Hoechst 33342 5 ml of stock solution 10 mgrml diluted in 500
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. ml of 2.9 wrv sodium citrate solution . The number of cells was counted after
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visualisation with an epifluorescent microscope Nikon Diaphot at 400 = magnifica- tion.
2.2.4. EÕaluation of spermatozoa by epifluorescent staining The viability and acrosome status of the semen used for IVF was evaluated using
Ž individual straws representing each ram and freezing treatment three straws per ram per
. treatment . The viability and acrosomal-integrity stains were performed on each straw.
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Ž . Viability live:dead ratio was assessed using a combination of propidium iodide PI
Ž and SYBR-14 fluorescent stains Molecular Probes, Leiden, The Netherlands; Garner et
. Ž
. al., 1986 . Straws were thawed
708C for 8 s and the contents re-suspended in
skim-milk, made up to 2.0 ml and then vortexed. A 200-ml aliquot of the diluted Ž
. spermatozoa was pipetted into an eppendorf tube and stained with 5 ml PI 0.1 mgrml
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plus 2 ml SYBR-14 0.1 mgrml . The tube was incubated at 338C for 10 min after which 5 ml of the stained suspension was placed on a slide under a coverslip. Three
slides were prepared per straw to get a more precise estimate of the ratio of viable to Ž
non-viable spermatozoa. Spermatozoa were assessed by fluorescent microscopy Olym- .
pus BX 60 at an excitation wavelength of 492 nm and a magnification of 400 = . Ž
Acrosome integrity was assessed using FITC-conjugated peanut agglutinin PNA– .
FITC . Once the 200 ml aliquot was removed for the viability assessment, the remaining Ž
. diluted spermatozoa were centrifuged 350 = g for 5 min and the supernatant was
discarded. The spermatozoa pellet was re-suspended in 2 ml PBS. The re-suspended Ž
. solution 500 ml was added to 500 ml of 100 ethanol and a 100-ml aliquot was added
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to 10 ml of PNA–FITC 1 mgrml and incubated for 30 min at room temperature. The spermatozoa were re-centrifuged at 350 g for 3 min to remove excess background stain,
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the spermatozoa pellet was re-suspended in 1.5 ml PBS and a sample 10 ml was placed Ž
. on a slide and air-dried Mortimer et al., 1987 . The pattern of PNA binding to the
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acrosomal membranes was assessed under fluorescent microscopy Olympus BX 60 at Ž
. excitation wavelength 492 nm 100 = magnification-oil immersion . A minimum of 100
cells was assessed for each thawed sample. 2.3. Experiment 2
This experiment was designed to establish the conception rate following both cervical and intrauterine insemination of frozen-thawed semen from the same bank of semen
used in Experiment 1. The fertilisation capacity in vivo was determined by intrauterine insemination of superovulated ewes. Semen from four rams that had shown significant
differences in IVF rate were used to test if this difference was evident in vivo.
2.3.1. Pregnancy rate after cerÕical and intrauterine insemination The effects of intrauterine versus cervical insemination with ‘‘slow’’- or ‘‘fast’’-frozen
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semen were investigated using a 2 = 2 factorial design. Mature ewes n s 119 were Ž
synchronised using a 12-day progestagen-impregnated vaginal pessary 30 mg fluroge- .
stone acetate — Chronogest, Intervet Laboratories, Cambridge, UK followed by a Ž
single intramuscular injection of 500 i.u. pregnant mare serum gonadotrophin PMSG, .
Intervet at pessary removal in late August. Sixty ewes were inseminated cervically at 55–57 h post sponge removal. Ewes were
inseminated standing on a raised platform at near eye-level to the inseminator so as to Ž
. Ž
. avoid undue stress to the animals Doney et al., 1976 . Semen was thawed 708C for 8 s
and an inseminate dose of approximately 100 = 10
6
spermatozoa was deposited as far as possible into the first fold of the cervix. The remaining ewes were used for intrauterine
insemination, by laparoscopy at 58–59 h post sponge removal. The procedure for Ž
. intrauterine insemination was similar to that described by Armstrong and Evans 1984 .
Two ewes were inseminated with the contents of each straw. Following thawing, each straw, which contained approximately 0.2 ml of semen, was diluted with an equal
volume of skim milk held in a water bath at 328C. An inseminate dose of 0.1 ml of
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6
. diluted semen approx. 25 = 10
spermatozoa was injected into each uterine horn. Raddled, vasectomised rams were introduced 10 days after insemination to check for
any repeats and ewes that were unmarked were assumed to be pregnant. These animals were slaughtered 30 days after insemination, reproductive tracts were recovered, the
numbers of corpora lutea and foetuses were recorded, and all foetuses were weighed.
2.3.2. Fertilisation rate in ÕiÕo Forty-seven mature ewes received the progestogen treatment described above and
Ž also received six intramuscular injections, each containing 34 mg FSH Folltropin-V,
. Vetrepharm, Ontario, Canada , 10–14 h apart, commencing 48 h before sponge removal
and ending 30 h before insemination. Insemination was by laparoscopy at 46 h post pessary withdrawal. Ewes were slaughtered on day 5 post insemination and the numbers
of corpora lutea and large follicles on each ovary were recorded. Each uterine horn was flushed through the oviduct with approximately 20 ml PBS supplemented with 3 BSA.
The flushings were searched for embryos and the embryos were graded according to recognised procedures.
2.4. Statistical analysis Data from Experiment 1 for the incidence of cleavage and blastocyst formation were
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analysed using the GENMOD procedure of SAS 1996 to fit a generalised linear model with a logit link function. Information on the number of cells per blastocyst and on the
results from staining were assessed by least squares analysis of variance using the GLM Ž
. procedure of SAS 1996 . In all cases, the models employed had terms for replicate,
treatment and individual ram. Data from Experiment 2 on conception rate were analysed using the GENMOD procedure and foetal weights were analysed by ordinary least
squares. The incidence of fertilisation and oocyte recovery were analysed using the Wilcoxon rank test with the individual animal as the experimental unit. A significance
level of 0.05 was used for testing effects in the models.
3. Results
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