Ability of frozen-thawed ram spermatozoa to bind to vitrified homologous oocytes

This study was designed to evaluate the ability of ram spermatozoa (thawed or thawed swim-up selected) to bind to homologous oocytes subjected to different treatments (in vitro matured or immature, fresh or vitrified). Frozen semen from Guirra breed males was tested on 1,267 oocytes harvested from pre-pubertal ewes at an abattoir. Highest proportions of oocytes with bound spermatozoa were recorded when in vitro matured oocytes, whether fresh or vitrified, were coincubated with thawed spermatozoa separated by swim-up (60.8 and 56.8% for fresh and vitrified matured oocytes, versus 9.4 and 11.1% for fresh and vitrified immature oocytes, respectively, p < 0.05). The number of bound spermatozoa per oocyte was also significantly higher for the swim-up selected spermatozoa than the thawed untreated spermatozoa (3.2 ± 0.5 vs 1.6 ± 0.7, p < 0.05, respectively), and higher still when fresh in vitro matured oocytes were used (8.3 ± 0.5).


Introduction
The Guirra (Sudat or Roja Levantina) sheep is the only ovine breed autochthonous to the Comunidad Valenciana, its current census being 3,640 females and 174 males.According to the FAO catalogue and Royal Decree 1682/97 (BOE, 1997), this breed is in danger of extinction and its recent decline has prompted a conservation program based on genetic resources which, among other measures, includes the creation of a semen bank.
The natural resistance of spermatozoa to cryopreservation varies among species, breeds and even among individual animals of the same breed.Detecting reduced-fertility males can be simple since these animals often show signs of altered semen quality that can be detected by routine analyses.However, not all the ejaculates showing adequate quality indicators are of normal fertility.The factors most commonly used to evaluate sperm quality in semen samples are sperm motility and morphology.Recently developed image
In the sheep, it has not been possible to significantly relate sperm motility characteristics with in vivo fertility (Sanchez-Partida et al., 1999), and contradictory results have been observed in cattle.Thus, while Budworth et al. (1988) and Bailey et al. (1994) found no significant correlations with in vivo fertility, Farrell et al. (1998) were able to detect some associations by including several of the variables obtained using the CASA system in the analysis (r 2 = 0.95).Other studies have been able to relate sperm abnormalities in bull ejaculates with both in vivo and in vitro fertility.
Besides morphological and motility tests, the recently developed in vitro techniques evaluate the functionality of the plasma membrane (endosmosis and acrosome exocytosis induction tests) or gamete interaction.Hence, in cattle, several authors have found significant relationships between fertility (rate of non-return to oestrous) and the percentage of endosmosis positive thawed spermatozoa, and between fertility and in vitro fertilisation or embryo development.
The aim of the present study was to evaluate the capacity of spermatozoa (thawed or swim-up prepared) to bind to fresh or vitrified oocytes that had been matured in vitro or left immature.Our ultimate goal is to include data related to sperm adhesion capacity, along with the data usually used, in a multiple regression analysis as a predictor of fertility.

Material and Methods
The ovaries used were obtained from ewes slaughtered at a commercial abattoir that were mainly of the Manchega breed and approximately 3 months of age.The ovaries were transported to the laboratory in a vacuum flask within 2 h of animal sacrifice.Cumulusoocyte complexes (COC) were recovered from the ovaries by aspirating follicles larger than 1 mm.Optimal quality oocytes were defined according to the criteria described by Martino et al. (1994Martino et al. ( , 1995) ) for the goat.The COCs were washed three times in PBS modif ied (PBSm) by supplementing with 0.5 g l -1 BSA, 36 mg l -1 sodium pyruvate, 1 g l -1 glucose and 50 µg ml -1 gentamicin.In total, 1,267 COCs were recovered and assigned to the following experimental groups: (1) fresh, immature; (2) fresh, in vitro matured; (3) vitrified, immature and (4) vitrified, in vitro matured.
Cumulus-oocyte complexes were vitrified according to the method described by Vajta et al. (1998).Exposure to the cryoprotectant solution was performed in two steps: in the first, the COCs were immersed in 10% ethylene glycol (EG), 10% dimethylsulfoxide (DMSO) in PBS for 45 s, and in the second step, in a solution containing 20% EG, 20% DMSO plus 0.5 M sucrose in PBS for 25 s.The COCs were then loaded in 0.25 ml straws and plunged into liquid nitrogen.Thawing was performed in a water bath at 20ºC after removing the cryoprotectant in three stages in Nunc ® plates containing 5 ml of medium: first in PBSm, 20% foetal bovine serum (FBS) and 0.25 M sucrose for 1 min; second in PBSm, 20% FBS and 0.15 M sucrose for 5 min and, in a final step, the COCs were washed in TCM199 medium supplemented with 10% FBS.
The COCs allotted to groups 2 and 4 were placed in culture for in vitro maturation in Nunc ® 4-well plates containing 500 µl of TCM199 medium (Tissue Culture Medium, Sigma M-4530) supplemented with 10% FBS, 10 ng ml -1 epithelial growth factor and 50 mg ml -1 gentamicin for 26 h at 38.5ºC in a 5%CO 2 water saturated atmosphere.For the sperm adhesion tests, the COCs were transferred to Nunc ® 4-well plates containing TALP (Tyrode-albumin-sodium lactate-sodium piruvate) supplemented with 10 µg ml -1 heparin.
The semen used for the tests was a pooled sample from 5 Guirra breed rams from a herd belonging to the Universidad Politécnica de Valencia.The ejaculates were obtained by the artificial vagina method, mixed (to avoid inter-individual differences) and cryopreserved by directly adding the diluent (Biociphos plus ® , IMV Technologies) to the ejaculate fraction at room temperature to obtain a 1:5 dilution.Next, the samples were loaded into 0.25 ml straws, during approximately 10 min.The straws were kept at 5ºC for 2 h.After this period, the semen was frozen at -120ºC in a nitrogen gas column (5 cm liquid nitrogen surface) for 10 min and finally stored in liquid nitrogen.The semen was thawed in a water bath at 42ºC for 12 s.
Once thawed, the semen was washed in STL medium (HEPES buffered TALP medium) and divided into two portions: one subjected to swim-up (Parrish et al., 1984) and the other preserved in an incubator at 37ºC.The concentration of semen used was 2 × 10 6 ml -1 and cocultures were maintained for 6 h at 38.5ºC in a 5% CO 2 water saturated atmosphere.
After coculture, the COCs were transferred to an Eppendorf tube containing sodium citrate and intensely vortexed for 3 min.Once denuded, the oocytes we-re stained for 5 min with Hoechst 33342 (Sigma, B-2261).Adhered spermatozoa were then counted under a fluorescence microscope (Nikon Eclipse E400, magnification 100×-400×).

Statistical analysis
The proportions of oocytes with adhered spermatozoa (thawed or thawed/swim-up prepared) according to the type of oocyte (fresh immature/in vitro matured or vitrified immature/in vitro matured) were compared by the chi-squared test, while the effect of the type of semen or oocyte and their interaction on the number of spermatozoa bound to each oocyte was evaluated using a general linear model (GLM, Statgraphics 4.1).

Results
Tables 1 and 2 show the results obtained in terms of the proportion of oocytes with bound spermatozoa and the number of bound spermatozoa per oocyte.Significant differences in the percentage of oocytes with adhered spermatozoa were observed between vitrified or fresh in vitro matured versus the vitrified or fresh immature oocytes (38.4% or 44.2% vs 8.5% or 16.3%, P < 0.05 respectively, Table 1).The two types of semen also yielded different results; a higher percentage of oocytes with adhered spermatozoa being recorded when the semen was swim-up selected after thawing (36.3% vs 13.6%, P < 0.05, Table 1).Best results in terms of percentage binding were obtained when matured vitrified or non-vitrified oocytes were coincu-bated with thawed swim-up prepared spermatozoa (56.8% or 60.8% respectively, Table 1).
Table 2 provides the numbers of spermatozoa adhered per oocyte.Both the type of semen and oocyte and interactions between them affected the sperm binding capacity.The spermatozoa selected by the swimup procedure after thawing showed a greater binding capacity than spermatozoa that had been simply thawed (3.2 ± 0.5 vs 1.6 ± 0.7, respectively, P < 0.05).The highest number of adhered prepared spermatozoa was recorded for the non vitrified matured oocytes compared to the other oocyte types (5.0 ± 0.5 vs 1.1 ± 0.9 to 1.9 ± 0.8, Table 2).

Discussion
Routine semen quality tests used to evaluate males only assess the spermatozoon.The importance of including tests in which spermatozoa and live oocytes interact is the information provided on fertilising capacity (Bavister, 1990).These tests also more closely mimic the steps occurring during fertilisation.The sperm binding test used by Zhang et al. (1998) enabled them to predict the fertilising capacity of semen from young high fertility bulls using three frozen ejaculates.This capacity was subsequently correlated (Zhang et al., 1999) with post-artificial insemination fertility (r = 0.88, p < 0.001).
oocytes (Zhang et al., 1995).Variation in the binding capacity of spermatozoa to individual oocytes is the most significant limiting factor for evaluating sperm binding capacity to the zona pellucida, since the characteristics of the zona pellucida are related to oocyte quality (Fazeli et al., 1996).To reduce inter-oocyte variability and conduct the semen evaluation test irrespective of the time of semen or oocyte availability requires the use of simple, cryopreservation protocols.
In our study, once the frozen semen had been thawed, the swim-up procedure served to select the most motile and viable sperm in the sample, explaining the improved results observed both in terms of the proportions of oocytes with bound spermatozoa and the number of spermatozoa per oocyte, compared to the directly thawed sperm.This selection technique has been previously related to an increased fertilisation rate (Parrish et al., 1984).
The effects of cryopreservation on oocytes include chromosome alterations (cell picnosis), reduced fertility rates and a high incidence of polyspermia.Cryopreservation causes damage to several organelles (microvilli, cortical granules, mitochondrial membranes) and the zona pellucida (Fuku et al., 1995).Here, vitrification was not found to be a determining factor for the percentage of oocytes with adhered spermatozoa, and this role was, instead, taken on by the factor in vitro maturation.The percentage of oocytes with adhered spermatozoa, irrespective of whether the oocytes had been vitrified or not, showed a drop of around 30% if the oocytes had not been matured in vitro.Maturation systems are not without their defects, and the overall efficiency of the process is still low for most species.It is thus common to compare the use of immature and mature oocytes.Our findings contradict those reported by other authors for different species (rabbit: Mocé and Vicente, 2001;pig: Martínez et al., 1993;cow: Niwa et al., 1991), who noted no binding differences between immature and in vitro matured oocytes.Low immature oocyte penetration rates have been attributed to quantitative and qualitative differences in the thickness and composition of the immature zona pellucida (Lucas et al., 2003).The zona pellucida forms during the maturation process (Braw-Tal and Yossefi, 1997) and it seems that immature oocytes differ from mature oocytes in the glycoproteins (Zp1, Zp2 and Zp3) of the zona pellucida, along with a lower number of spermatozoon receptors (Lucas et al., 2003).
The decreased number of spermatozoa adhered to vitrified in vitro matured oocytes compared to oocy-tes subjected to in vitro maturation immediately after their recovery could be explained by alterations to the zona pellucida induced by rupture of the cortical granules during cryopreservation, leading to the early permeabilization of the zona pellucida (Van Blerkom, 1991;Vincent and Johnson, 1992).Enzymes released in the cortical region modify sperm receptors and components of the vitelline membrane, preventing sperm binding (Otoi et al., 1993;Fuku et al., 1995;Martino et al., 1996).
In summary, our findings suggest the suitability of using the protocol described by Vajta et al. (1998) to vitrify immature oocytes obtained from abattoir sheep.These vitrified oocytes could be used to create a bank for sperm binding tests after subjecting the oocytes to 24 h of in vitro maturation.

Table 1 .
Percentage of oocytes with bound spermatozoa