Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro

Animal Reproduction Science 62 Ž2000. 265–275 www.elsevier.comrlocateranireprosci

Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro G.P. Byrne a , P. Lonergan a,) , M. Wade a , P. Duffy a , A. Donovan b, J.P. Hanrahan b, M.P. Boland a a

Department of Animal Science and Production, UniÕersity College Dublin, Lyons Research Farm, Newcastle, Dublin, Ireland b Teagasc Athenry Research Centre, Athenry, Galway, Ireland Received 24 August 1999; received in revised form 7 February 2000; accepted 23 February 2000

Abstract Ram spermatozoa are most susceptible to damage during freezing between the temperatures of y108C and y258C. The objectives of the present study were to examine how freezing rate through this critical temperature zone affected the fertility of spermatozoa as assessed in vivo and in vitro. Semen from six adult rams was frozen at two different rates Ž‘‘fast’’: 58Crmin from q5 to y258C; ‘‘slow’’: 0.58Crmin from q5 to y258C.. In Experiment 1, semen from the fast and slow treatments was used to fertilize ovine oocytes that had been matured in vitro. Semen from the fast treatment yielded a higher cleavage rate Ž57% vs. 26%; P - 0.001. and more blastocysts per oocyte Ž28% vs. 13%, P - 0.001. than slow-frozen. No correlation was found between fertilizing ability and viability as assessed by fluorescent probes. Experiment 2 was designed to establish the conception rates following both cervical and intrauterine insemination of frozen-thawed semen from the same bank of semen as used in Experiment 1. Ewes were superovulated with FSH and inseminated by laparoscopy with frozen semen. A significant difference was found in the number of fertilized ova following embryo recovery Ž81.4% vs. 39.3%; P - 0.001.. In a further study, 119 mature cull ewes were inseminated following a 12-day synchronization treatment with frozen semen by either intrauterine Žlaparoscopic. or cervical insemination. Insemination with fast-frozen semen resulted in a significantly higher pregnancy rate Ž P - 0.05. irrespective of method of insemination. The data show that freezing rate affects the proportion of spermatozoa that retain their fertilizing ability post-thawing. However, once fertilization has occurred, development to the blastocyst stage is independent of freezing rate. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Sheep male reproduction; Spermatozoa; Embryo; IVF; Superovulation; Cryopreservation

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Corresponding author. Tel.: q353-1-6288385; fax: q353-1-6288421. E-mail address: [email protected] ŽP. Lonergan..

0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 1 2 1 - 4

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1. Introduction Freezing mammalian spermatozoa offers many advantages to the livestock industry, particularly in conjunction with genetic evaluation and selection programmes, such as in sire reference schemes ŽMaxwell, 1984.. However, the biggest obstacle to the exploitation 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 damage to membrane structures ŽQuinn 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 the insemination dose ŽWatson, 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 nitrogen ŽMazur, 1965.. In the case of ram spermatozoa, most damage occurs between y108C and y258C; the region of ice crystallisation ŽSalamon 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 ŽWatson, 1995.. There are some reports in the literature dealing with the effects of freezing rate on post-thaw motility and acrosome integrity ŽWatson and Martin, 1975; Fiser and Fairfull, 1986.. O’Neill Ž1998. observed that semen frozen rapidly Žfrom 58C to y258C at y58Crmin. had significantly better viability, mitochondrial activity and acrosome integrity than after a slower Žy0.58Crmin. 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 O’Neill Ž1998., 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 fertility. Following collection, motility Žwave motion. was examined and graded on a scale of 0–5 Ž0 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 spermatozoa in the sample was assessed using a previously calibrated colorimeter ŽEEL, Halstead, Essex.. Semen was diluted up to 7.5 ml with an extender, based on skim milk

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and egg yolk, which was maintained at 358C. The samples were then placed in a cold room Ž58C. 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 diluted samples were placed in a refrigerated Ž58C. centrifuge ŽSorvall 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 then loaded into 0.25-ml straws ŽMinitub, 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 freezer ŽPlanar 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 Chemicals were obtained from Sigma ŽSt. Louis, MO, USA. unless otherwise indicated. The conditions for maturation were a slightly modified version of those described by Lonergan et al. Ž1996.. Briefly, a stock solution of epidermal growth factor ŽEGF. was prepared Ž10 mgrml. in medium 199 ŽM199. and stored at y208C until use, at which time it was diluted to a working concentration of 10 ngrml. Ovaries were collected from slaughtered ewes Žduring the breeding season. and placed in phosphatebuffered saline ŽPBS. at approximately 308C for transport to the laboratory. After washing in fresh PBS, cumulus oocyte complexes ŽCOCs. 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 stereomicroscope and passed through two washes of aspiration medium followed by three washes in modified PBS Žsupplemented with pyruvate Ž36 mgrml., gentamycin Ž50 mgrml. and bovine serum albumin Ž0.5 mgrml; Sigma, fraction V, cat. a A-9647.. Groups of COCs were then transferred into four-well plates Ž50 COCs per well, Nunc, Roskilde, Denmark. containing 500 ml of maturation medium ŽM199q 10 ngrml EGF q 10% fetal calf serum. and incubated for 24 h in 5% CO 2 in humidified air at 398C. 2.2.2. In Õitro fertilisation For IVF, matured COCs were washed four times in PBS and twice in synthetic oviduct fluid medium ŽSOFB; O’Brien et al., 1997.. Oocytes were then transferred into four-well plates containing 250 ml of SOFB medium per well Ž50 oocytes per well.. For sperm preparation, three straws, representing one ram and a particular treatment, were thawed Ž708C 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 obtained by centrifugation, at 700 = g, on a Percoll ŽPharmacia, Uppsala, Sweden. discontinuous density gradient Ž2 ml of 45% Percoll over 2 ml of 90% Percoll. for 20

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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 2 in 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 humidified atmosphere of 5% CO 2 , 5% O 2 and 90% N2 at 398C ŽCarolan et al., 1995.. 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 of 20–30, into culture droplets Žone zygote per microliter of medium.. Fetal calf serum ŽFCS; 10% vrv. was added to each droplet 24 h after placement in culture Ži.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 and then stained using Hoechst 33342 Ž5 ml of stock solution Ž10 mgrml. diluted in 500 ml of 2.9% Žwrv. sodium citrate solution.. The number of cells was counted after visualisation with an epifluorescent microscope ŽNikon Diaphot. at 400 = magnification. 2.2.4. EÕaluation of spermatozoa by epifluorescent staining The viability and acrosome status of the semen used for IVF was evaluated using individual straws representing each ram and freezing treatment Žthree straws per ram per treatment.. The viability and acrosomal-integrity stains were performed on each straw. Viability Žlive:dead ratio. was assessed using a combination of propidium iodide ŽPI. and SYBR-14 fluorescent stains ŽMolecular Probes, Leiden, The Netherlands; Garner et al., 1986.. Straws were thawed Ž708C 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 spermatozoa was pipetted into an eppendorf tube and stained with 5 ml PI Ž0.1 mgrml. plus 2 ml SYBR-14 Ž0.1 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 non-viable spermatozoa. Spermatozoa were assessed by fluorescent microscopy ŽOlympus BX 60. at an excitation wavelength of 492 nm and a magnification of 400 = . Acrosome integrity was assessed using FITC-conjugated peanut agglutinin ŽPNA– FITC.. Once the 200 ml aliquot was removed for the viability assessment, the remaining diluted spermatozoa were centrifuged Ž350 = g for 5 min. and the supernatant was

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discarded. The spermatozoa pellet was re-suspended in 2 ml PBS. The re-suspended solution Ž500 ml. was added to 500 ml of 100% ethanol and a 100-ml aliquot was added to 10 ml of PNA–FITC Ž1 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, the spermatozoa pellet was re-suspended in 1.5 ml PBS and a sample Ž10 ml. was placed on a slide and air-dried ŽMortimer et al., 1987.. The pattern of PNA binding to the acrosomal membranes was assessed under fluorescent microscopy ŽOlympus BX 60. at excitation wavelength 492 nm Ž100 = 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 semen were investigated using a 2 = 2 factorial design. Mature ewes Ž n s 119. were synchronised using a 12-day progestagen-impregnated vaginal pessary Ž30 mg flurogestone acetate — Chronogest, Intervet Laboratories, Cambridge, UK. followed by a single intramuscular injection of 500 i.u. pregnant mare serum gonadotrophin ŽPMSG, 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 avoid undue stress to the animals ŽDoney et al., 1976.. Semen was thawed Ž708C 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 intrauterine insemination was similar to that described by Armstrong and Evans Ž1984.. 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 diluted semen Žapprox. 25 = 10 6 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 also received six intramuscular injections, each containing 34 mg FSH ŽFolltropin-V,

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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 analysed using the GENMOD procedure of SAS Ž1996. 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 procedure of SAS Ž1996.. 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 3.1. Experiment 1 The proportions of oocytes, averaged over all replicates, fertilised Žcleaved. and the proportions undergoing development to the blastocyst stage are presented in Table 1. Overall, insemination with sperm frozen using the ‘‘fast’’ freeze protocol produced a significantly higher cleavage rate compared with the ‘‘slow’’ freeze Ž57% vs. 26%, P - 0.001.. Insemination with ‘‘fast’’-frozen semen produced more than double the percentage of blastocysts from oocytes than the slow freeze Ž P - 0.001. based on oocytes in culture. However, when expressed as a percentage of cleaved oocytes, there was no effect of freezing rate on developmental competence.

Table 1 The effect of freezing rate on the incidence of fertilisation and blastocyst production from sheep oocytes following maturation, fertilization and culture in vitro Values, within columns, with different superscripts are significantly different Ž P - 0.001.. Freezing rate

No. of oocytes

Cleavage percentage

Blastocyst yield Ž%. Total

Fast Slow

899 845

a

57 26 b

a

28 13 b

From cleaved 49 51

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Table 2 Percentages of acrosome-intact, acrosome-damaged and acrosome-reacted ram spermatozoa following postthaw analysis using the fluorescent probe PNA-FITC Žleast squares means"S.E.. No significant difference. Treatment

Acrosome-intact

Acrosome-damaged

Acrosome-reacted

Fast frozen Slow frozen

45.7"4.14 49.0"4.14

39.7"2.34 36.0"2.34

14.7"3.39 15.0"3.39

There were highly significant differences among rams in the fertilising ability of their frozen-thawed semen Ž P - 0.001.. Fertilisation rate ranged from 36% to 84% following insemination with semen frozen by the fast freezing curve, and between 7% and 79% using semen frozen at a slow rate. For five of the six rams used in the experiment, there was a clear difference in the rate of cleavage in favour of fast freezing; however, one ram had similar fertilisation rates following insemination with semen from either freeze Ž70% fast, 79% slow.. This was reflected in a highly significant ram = freeze interaction Ž P - 0.001.. There was also a significant replicate= freeze interaction Ž P - 0.001. that reflected variation in the magnitude of the advantage of ‘‘fast’’ freezing among replicates rather than any reversal of ranking of the freezing treatments. Freezing rate had no significant effect on the number of cells per blastocyst. The least squares means for cell number were 126 " 12.4 Ž n s 256. and 141 " 13.0 Ž n s 111. for the ‘‘fast’’ and ‘‘slow’’ treatments, respectively. There was no significant difference between slow and fast freezing with respect to the viability of frozen-thawed ram spermatozoa as assessed by epifluorescent staining. However, there was significant ram-to-ram variation in percentage live spermatozoa post-thaw Ž P - 0.01.. Similarly, freezing regime had no significant effect on the percentage acrosome intact, damaged or reacted spermatozoa following post-thaw analysis ŽTable 2.. There was no relationship between ram effects and the percentage viable sperm or between ram effects and IVF results. 3.2. Experiment 2 When the teaser rams were removed, 35 ewes remained unmarked, and 33 of these were pregnant at slaughter. Method of insemination had a highly significant effect on pregnancy rate Ž P - 0.001.. There was a significant effect of freezing rate on pregnancy

Table 3 Effect of freezing rate and method of insemination on pregnancy in ewes Freezing rate

Site of insemination

No. of ewes

Percentage of pregnant ewes

No. of foetuses

Fast Slow Fast Slow

Uterine Uterine Cervical Cervical

30 29 30 30

60 40 13 0

28 17 4 –

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Table 4 Ovulation rate, embryo recovery and fertilisation for superovulated ewes inseminated into the uterine lumen using two frozen-thawed semen types Values, within rows, with different superscripts are significantly different Ž P - 0.001.. Freezing rate Fast

Slow

No. of ewes: Inseminated Ovulated Yielded ovarembryos Yielded embryos Mean ovulation rate

23 22 18 18 11.4

24 22 21 14 7.5

No. of oocytes: Recovered Fertilised

95 Ž36%. 71 Ž81.4%. a

118 Ž73.6%. 49 Ž39.3. b

rate Ž P - 0.01., with 22 of the pregnant animals having been inseminated with fast-frozen semen ŽTable 3.. Rate of freezing had no significant effect on foetal weight and there was no individual ram effect on this trait. The results from the evaluation of fertilisation rate in vivo using superovulated ewes are given in Table 4. The higher mean ovulation rate in the ‘‘fast’’ group is attributable to two animals having in excess of 20 ovulations each. Freezing rate had a highly significant Ž P - 0.001. effect on the number of ewes that yielded fertilised eggs. In the fast freeze group, all ewes that ovulated and yielded oocytes on flushing Ž18r22. had some ova fertilised. Of the ewes on the slow freeze treatment that ovulated and yielded ova on recovery Ž21r22., seven had all oocytes unfertilised. There was a significant freezing effect on the fertilisation rate of recovered ova Ž81.4% for fast freezing compared with 39.3% after slow freezing; P - 0.01.. Data in Table 5 summarise the developmental stages of the embryos following recovery. There was no effect of freezing rate on the proportion of cleaved oocytes that proceeded to the morularblastocyst stage. There was no significant difference between individual rams in terms of their fertilising ability. Table 5 Stages of embryonic development following laparoscopic intrauterine insemination in superovulated ewes MrB: morulaerblastocysts expressed over total oocytes recovered. Values, within rows, with different superscripts are significantly different Ž P - 0.001.. Freezing rate

Slow Fast a

No. of fertilized oocytes

49 71

Cell stage attained 2–8 cells

8–16 cells

Morula

Blastocyst

2 10

13 5

34 51

0 5

Expressed as a percentage of fertilized oocytes.

Percentage MrB

Percentage MrBa

29 Ž34r118. a 59 Ž56r95. b

69 Ž34r49. 79 Ž56r71.

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4. Discussion The results of this study provide clear evidence that the freezing rate used through the critical window of damage influences the fertility of spermatozoa following insemination in vitro and in vivo. The overall proportion of oocytes that yielded blastocysts in vitro for the ‘‘fast’’ freeze Ž49%. compare favourably with sheep IVF data from other research groups using frozen semen ŽGuerin et al., 1992; O’Brien et al., 1997. and are similar to those reported for cattle in our laboratory ŽLonergan et al., 1999.. There were significant differences in overall incidences of cleavage Ž57% vs. 26%. and blastocyst rate Ž28% vs. 13%. between the two freezing curves. However, when blastocyst rates are expressed as a percentage of cleaved oocytes, there was no effect of freezing rate Ž49% vs. 51%.. This suggests that irrespective of freezing treatment, once cleaved, oocytes were equally competent to develop to the blastocyst stage. This would support the suggestion of O’Neill Ž1998. that a fast freezing rate to y258C would improve the fertility of frozen-thawed ram semen. According to Watson Ž1995., slow dehydration of spermatozoa is associated with cell survival, whereas at more rapid dehydration rates, cell death is more likely. In reality, our ‘‘fast’’ freeze is no more than an optimal freezing rate and our ‘‘slow’’ freeze a sub-optimal freezing rate through the critical temperature zone of y158C to y258C, where cell damage is most likely to occur ŽSalamon and Maxwell, 1995.. Reducing the temperature at a rate of y58Crmin through this critical region appears to be close to optimal in achieving highest rates of cleavage. Seigneurin and Blesbois Ž1995. cryopreserved rooster spermatozoa from q58C to y358C at rates of y1, y5, y7, y10 and y158Crmin. The highest fertilisation rates following artificial insemination were obtained from semen frozen at y58C or y78C. This result would agree with Mazur Ž1965., who suggested that spermatozoa frozen at sub-optimal temperatures are exposed too long to ‘‘solution effects’’. The highly significant individual ram differences in sperm viability and fertilisation success are not new. Fukui et al. Ž1988. using fresh semen and Morris et al. Ž1998. using frozen-thawed semen demonstrated that individual rams affected not only the in-vitro fertilisation rate, but also the ability of the cleaved oocyte to develop after fertilisation. This phenomenon has also been reported for bull spermatozoa ŽIritani et al., 1986.. With fast freezing, variation in cleavage rates ranged from 36% to 84%. An even greater spread is evident in the slow freeze Ž7–79%.. The fact that the difference in fertilisation rates is quite large in five of the six rams when such a difference did not exist following epifluorescent staining is alarming. The number of cells per blastocyst is a potential indicator of the quality of the developing embryo. The absence of any effect of freezing rate on cell number is consistent with the fact that the proportion of cleaved oocytes that proceeded to the blastocyst stage was also unaffected by freezing rate. The results from semen analysis show that there were no significant differences between the two freezing curves in terms of viability post-thaw, although there were significant differences between rams. Freezing at the slow rate appears to induce a semi-lethal change in the sperm in that while the thawed sample is morphologically ‘‘ viable’’ as observed by epifluorescent staining, its fertilising ability is compromised.

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The lack of correlation between the fluorescent staining data and the results of IVF are perhaps not surprising when one considers that for IVF, a highly motile sample were selected by Percoll centrifugation. An important aspect in evaluating the quality of a semen sample is the ability of spermatozoa to undergo the acrosome reaction. In this study, there were no differences between the freezing curves in terms of acrosome intact, damaged and reacted cells in any of the thawed samples. The process of freezing and thawing induced acrosomal damage Žeither partial or complete acrosome loss. in 43–57% of the sperm population. While only a small proportion of sperm were recorded as being acrosome-reacted, the proportion of spermatozoa with partial acrosome loss Ždamaged. must be discounted from taking part in fertilisation since they are likely to undergo complete acrosome reaction shortly after insemination. O’Neill Ž1998. found significant differences in viability between the two freezing curves but found no difference in acrosome integrity. It appears that freezing down to y258C using either freezing curve causes significantly less acrosome damage than freezing to y108C ŽO’Neill, 1998.. The significant difference between methods of insemination was no surprise since laparoscopic deposition of semen close to the site of ovulation will increase the chance of fertilisation occurring and agrees with the findings of other research groups Žreviewed by Gordon, 1997.. The observation that freezing rate had a significant effect on pregnancy rate is consistent with the conclusions drawn from the in vitro results ŽExperiment 1. and demonstrates the utility of the IVF procedure for evaluation of frozenrthawed semen. Four of 30 ewes inseminated cervically with ‘‘fast’’ semen were pregnant. The poor pregnancy rates were almost certainly a consequence of the low sperm numbers per straw. It is not surprising that no difference in pregnancy rate was observed in those inseminated by laparoscopy since the sperm populations have much less distance to travel to reach the site of fertilisation. In Experiment 1, there was no difference in the cell numbers of blastocysts produced from either slow or fast freezing and the conclusion drawn was that once cleavage occurred, either group had equal opportunity of developing to the blastocyst stage. The results of the foetal weights provide more convincing proof of this. It is reasonable to suggest that the rigours of sub-optimal freezing renders most of the sperm population unable to take part in the fertilisation process; however, those that survive the freezing are as competent of undergoing fertilisation as those in the fast freeze. Insemination by laparoscopy, while ensuring fertility, is associated with a reduction in ovum recovery in superovulated ewes. Armstrong and Evans Ž1984. observed that the ovum recovery rate was as much as 15% lower following intrauterine than cervical insemination. In the in vitro experiment, the overall difference in cleavage rate between the two frozen semen types was over 30 percentage points Ž57% vs. 26%.. An even greater difference was observed Ž81% vs. 39%. following superovulation. In conclusion, the studies reported here have shown that IVF evaluation of frozenrthawed semen provides a valid assessment of the ability of the semen to achieve fertilisation in vivo and furthermore indicates that it is the ‘‘fertilisation’’ capacity of the semen that is compromised by slow freezing and not their ability to get to the site of fertilisation. The semen chosen for the in vivo work was from lots known to have a

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