Heterologous In Vitro Fertility Evaluation of Cryopreserved Iberian Red Deer Epididymal Spermatozoa with Zona-intact Sheep Oocytes and its Relationship with the Characteristics of Thawed Spermatozoa
Contents
A heterologous in vitro system, using zona-intact sheep oocytes, was used to evaluate the relationship between sperm factors of Iberian red deer thawed epididymal sperm and the percentage of cleaved oocytes. Epididymal spermatozoa were recovered from six males, diluted with freezing extender and cryopreserved. After thawing sperm motility (SM) and acrosome and membrane integrities were evaluated. Again, these parameters were assessed after incubation in freezing extender at 37°C for 2 h. After cryopreservation the values for SM and acrosome and membrane integrities were high (∼80, 80 and 70% respectively). However, these values significantly decreased after incubation (∼59, 62 and 47% respectively). Red deer thawed epididymal sperm fertilized zona-intact sheep oocytes, although the percentage of cleaved oocytes was low (∼22%). No relationship was found between sperm parameters assessed after thawing and the percentage of cleaved oocytes. Likewise, any sperm parameter evaluated after incubation was assessed in relation to the percentage of cleaved oocytes. However, acrosome and membrane integrities were near to significance (p = 0.06 and p = 0.09 respectively). Then, we conducted a reduced model with these two variables and both were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Thus, acrosome and membrane integrities were related to the percentage of cleaved oocytes negatively and positively respectively. It was concluded that the classical parameters assessed in deer thawed sperm samples can be good predictors of the ability to fertilize zona-intact sheep oocytes.
Introduction
Genome resource banks are very useful for preserving wild and endangered species. This interest in preserving germplasm of the wild species has resulted in greater attention paid to the possible recovery of sperm from the epididymides of dead animals. Thus, the postmortem collection constitutes the best source of germplasm in areas of regulated hunting. Such is the situation of the Iberian red deer (Cervus elaphus hispanicus), which is one of the most appreciated hunting species in Spain. The creation of a germoplasm bank would greatly enhance the management of these populations and preserve their genetic wealth, which is under threat by inbreeding and hybridization.
We have demonstrated that viable spermatozoa can be retrieved from deer epididymides maintained at room temperature (approximately 20°C) up to 24 h after death (Garde et al. 1998). In addition, we recently reported that viable spermatozoa could be recovered from deer epididymides stored at 5°C up to 4 days after death (Soler et al. 2003). Also, these sperm samples have high viability even after cryopreserving them (Soler et al. 2005). However, cryopreservation causes sperm damage, a reduction on sperm viability and functionality and a lower fertility (Watson 2000; Watson and Fuller 2001). Thus, before we use the frozen sperm to obtain offspring, it is necessary to understand the characteristics and viability of thawed sperm cells obtained from dead animals and to examine the most important factors that affect their fertilizing ability. Assessment of the sperm quality by the evaluation of standard semen parameters is a routine procedure in the prediction of male fertility. However, the best assessment of in vitro function involves sperm–oocyte interaction (Bavister 1990).
Heterologous in vitro fertilization (IVF) is an attractive method to evaluate the fertilizing capacity of sperm samples in wild species because it does not require the use of valuable homologous gametes. The cross-species fertilization of oocytes from domestic farm species has been proven successful, using cryopreserved sperm form deer (Comizzoli et al. 2001) and non-domestic Bovidae (McHugh and Rutledge 1998; Roth et al. 1999). Recently, the sperm function was evaluated in several endangered African antelope by means of a heterologous IVF system with zona-intact domestic cow oocytes (Kouba et al. 2001).
The success of heterologous IVF in several wild ungulates, as has been cited above, suggests that this system may be useful to test sperm function in a range of ungulates. Thus, the main purpose of the present study was to determine the relationship between sperm characteristics from Iberian red deer thawed epididymal spermatozoa and the ability to fertilize zona-intact sheep oocytes.
Materials and Methods
All chemicals were of reagent grade and were purchased from Sigma (St. Louis, MO, USA), Merck (Madrid, Spain), Gibco (Grand Island, NY, USA) and Minitüb (Tiefenbach, Germany).
Collection and assessment of epididymal spermatozoa
Sperm samples were obtained from six mature red deer stags that were legally culled and hunted in their natural habitat. The hunting of stags was performed in accordance with the harvest plan of each game reserve. The harvest plans were made following Spanish Harvest Regulation, Law 2/93 of Castilla-La Mancha, which conforms to European Union Regulation.
Scrota containing testes and epididymides were placed in plastic bags and transported to the laboratory at room temperature (approximately 20°C) within 2 h after being removed. The samples were processed as soon as they arrived at the laboratory. Spermatozoa were collected from epididymides as described by Soler et al. (2003). Briefly, spermatozoa were recovered from the distal portion of the epididymis by cutting the caudae epididymides with a surgical blade and collecting the oozing sperm mass and placing it in 1 ml of Triladyl® (Minitüb, Tiefenbach, Germany) with a 20% egg yolk (containing 6% glycerol).
After the spermatozoa collection and before refrigeration, a standard semen evaluation was made. Sperm concentrations of the original suspensions were determined, using a haematocytometer. In addition, sperm motility (SM) and acrosome integrity were assessed for each sample, as described below. The percentage of individual SM and acrosome integrity were estimated in the diluted samples.
When all the assessments were carried out, the sperm mass was again diluted at room temperature to a final sperm concentration of ∼400 × 106 sperm/ml with the Triladyl® medium.
Cryopreservation and assessment of frozen-thawed spermatozoa
The diluted sperm suspension in a 15-ml centrifuge tube (Afora, Iwaki, Japan) was placed in a beaker with water (75 ml at room temperature) and transferred to a refrigerator at 5°C. Diluted samples were refrigerated slowly at 5°C for 1 h, equilibrated at that temperature for 2 h and loaded into 0.25 ml straws (100 × 106 sperm/straw). The straws were frozen in nitrogen vapours, 4 cm above the surface of liquid nitrogen, for 10 min and then plunged into liquid nitrogen. The straws remained for a minimum period of 1 year in liquid nitrogen before the thawing was carried out.
Then, straws were thawed in a water bath at 37°C. After 5 min at this temperature subsamples were taken for the assessment of SM. Also, the acrosomal integrity was evaluated after a 1 : 10 dilution in a 2% glutaraldehyde in 0.165 m cacodylate/HCl buffer (pH 7.3). The percentage of spermatozoa with intact acrosomes (% NAR) was assessed by phase-contrast microscopy at ×400. The plasma membrane integrity was assessed, using an osmotic swelling test, as described by Garde et al. (1998). The osmotic swelling technique consisted of mixing 0.01 ml of diluted sperm samples with 0.1 ml of hypo-osmotic solution (100 mOsm/kg) and incubating the mixture at room temperature for 30 min. The samples were then fixed in a 2% glutaraldehyde buffered solution and evaluated by phase-contrast microscopy at ×400. The sperm membrane was considered intact in cases where the sperm tail was coiled (% HOST).
Thawed samples were incubated at 37°C for 120 min in the diluent. At the end of this incubation, sperm suspensions were again assessed for motility, acrosome integrity and membrane integrity as described above
The entire experiment was conducted three times; thus, independent straws of spermatozoa from each stag in triplicate were assessed for motility, the acrosome status and membrane integrity. Two hundred sperm cells were assessed in each sample and for each sperm evaluation technique.
Heterologous IVF
Domestic sheep ovaries were collected at an abattoir and transported to laboratory in saline (30°C) between 3 and 4 h after being removal. Immature oocytes were collected from ovaries using 19-gauge needle in TCM-199 supplemented with HEPES (2.39 mg/ml), heparin (2 μl/ml) and gentamycin (40 μg/ml). Aspirated cumulus oocyte complexes (COC) were washed in TCM-199 gentamycin and those with dark homogeneous cytoplasm and surrounded by tightly packed cumulus cells were selected and placed in four-well plates containing 500 μl of TCM-199 supplemented with cysteamine (100 μm) and the epidermal growth factor (10 ng/ml) and matured at 38.5°C under 5% CO2. After 24 h, COC were washed in synthetic oviduct fluid supplemented with essential and non-essential amino acids (Gardner et al. 1994) and cumulus cells were removed by gentle pipeting. Oocytes were transferred into four-well plates with 400 μl of SOF supplemented with 20% of oestrous sheep serum and 40 μg/ml gentamycin under mineral oil.
Thawed epidydimal spermatozoa were selected on a Percoll discontinuous density gradient (45/90%) and were capacitated in the fertilization medium with heparin (5 μg/ml) for 15 min. Sperm was co-incubated with oocytes at a final concentration of 2 × 106/ml at 38.5°C under 5% CO2. Oocytes were evaluated 40 h later for cleavage (two or more cells). The number of cleaved oocytes was assessed visually under an inverted microscope.
Heterologous IVF was carried out four times for each male and a minimum of 15 oocytes were used each time. Mean value of cleaved oocytes was noted.
Statistics
All variables were transformed to attain a normal distribution. Two multiple linear regression analyses were carried out where the dependent variable was the percentage of cleaved oocytes in both analyses and the independent variables were sperm parameters assessed at thawing and at incubation for each analysis.
Results
Sperm characteristics
Before cryopreservation the mean values for SM and acrosome integrity were 91.66 ± 1.66 and 91.83 ± 2.08 respectively. After thawing the mean value for SM was 80.27 ± 2.44% and this value decreased to 59.44 ± 3.72% after incubation at 37°C for 2 h. Acrosome and membrane integrities after cryopreservation were 80.33 ± 1.95 and 69.83 ± 3.68 with a lower value after incubation: 62.00 ± 3.70 and 47.16 ± 3.74 respectively. The individual values for each male and the mean values for all the sperm parameters assessed in the different times are shown in Tables 1, 2 and 3.
| Sperm parameters (%) | Male 1 | Male 2 | Male 3 | Male 4 | Male 5 | Male 6 | Mean |
|---|---|---|---|---|---|---|---|
| SM | 90 | 95 | 90 | 85 | 95 | 90 | 91.66 ± 1.66 |
| NAR | 87 | 95 | 89 | 96 | 98 | 86 | 91.83 ± 2.08 |
- SM, sperm motility; NAR, intact acrosome.
| Sperm parameters (%) | Male 1 | Male 2 | Male 3 | Male 4 | Male 5 | Male 6 | Mean |
|---|---|---|---|---|---|---|---|
| SM | 83.33 ± 1.66 | 86.67 ± 1.66 | 71.67 ± 8.81 | 78.33 ± 6.66 | 86.67 ± 1.66 | 75.00 ± 8.66 | 80.27 ± 2.44 |
| NAR | 81.33 ± 7.68 | 84.33 ± 5.04 | 76.33 ± 2.84 | 74.33 ± 7.35 | 82.33 ± 1.45 | 83.33 ± 2.02 | 80.33 ± 1.95 |
| HOST | 68.67 ± 5.78 | 86.00 ± 4.16 | 64.33 ± 8.21 | 69.67 ± 9.38 | 75 | 55.33 ± 9.06 | 69.83 ± 3.68 |
- SM, sperm motility; NAR, intact acrosome; HOST, membrane integrity.
| Sperm parameters (%) | Male 1 | Male 2 | Male 3 | Male 4 | Male 5 | Male 6 | Mean |
|---|---|---|---|---|---|---|---|
| SM | 58.33 ± 9.27 | 66.67 ± 11.66 | 56.67 ± 13.64 | 48.33 ± 8.33 | 63.33 ± 8.33 | 63.33 ± 6.66 | 59.44 ± 3.72 |
| NAR | 68.33 ± 2.72 | 65.00 ± 3.78 | 66.00 ± 9.01 | 56.33 ± 18.02 | 63.67 ± 10.74 | 52.67 ± 7.38 | 62.00 ± 3.70 |
| HOST | 53.33 ± 12.03 | 57.67 ± 7.21 | 50.00 ± 16.16 | 38.67 ± 6.17 | 38.67 ± 233 | 44.67 ± 7.68 | 47.16 ± 3.74 |
- SM, sperm motility; NAR, intact acrosome; HOST, membrane integrity.
Heterologous IVF
The mean value of cleaved oocytes for all males was 21.71 ± 3.12% with a range of ∼15–29% between males for all replicates (Table 4). Nevertheless, upto 55% of the oocytes were fertilized by some males in different replicates (data not shown).
| IVF Parameters | Male 1 | Male 2 | Male 3 | Male 4 | Male 5 | Male 6 | Mean |
|---|---|---|---|---|---|---|---|
| Oocytes number | 15.25 ± 0.62 | 17.25 ± 1.31 | 17.00 ± 1.47 | 20.00 ± 1.41 | 18.25 ± 0.94 | 20.75 ± 2.42 | 18.08 ± 0.65 |
| % cleaved | 21.25 ± 8.56 | 23.25 ± 6.60 | 18.50 ± 4.48 | 22.50 ± 11.99 | 15.25 ± 4.30 | 29.50 ± 10.57 | 21.71 ± 3.12 |
Relationship between sperm parameters and in vitro fertilization
We performed two multiple linear regressions including the percentage of cleaved oocytes as dependent variables in both analyses and as independent variables: SM, acrosome and membrane integrities evaluated after thawing for one analysis (Table 5) and the same sperm parameters assessed after incubation for the other (Table 6). The analysis carried out with sperm variables assessed after thawing rendered a full model not significant (F3,6 = 0.39; p = 0.77) and any sperm variable was related to the percentage of cleaved oocytes (Table 5).
| Independent variable | Coefficient | Standard error | t | p |
|---|---|---|---|---|
| Constant | 0.218 | 0.731 | 0.298 | 0.794 |
| SM | −0.462 | 0.851 | −0.543 | 0.642 |
| NAR | 0.727 | 0.845 | 0.861 | 0.480 |
| HOST | −0.027 | 0.493 | −0.055 | 0.961 |
- Total model R2 = 0.368. SM, sperm motility; NAR, intact acrosome; HOST, membrane integrity.
| Independent variable | Coefficient | Standard error | t | p |
|---|---|---|---|---|
| Constant | 1.009 | 0.257 | 3.924 | 0.059 |
| SM | −0.121 | 0.235 | −0.515 | 0.658 |
| NAR | −1.049 | 0.276 | −3.804 | 0.063 |
| HOST | 0.706 | 0.240 | 2.939 | 0.099 |
- Total Model R2 = 0.885. SM, sperm motility; NAR, intact acrosome; HOST, membrane integrity.
Likewise, all sperm parameters evaluated after incubation were not related with the percentage of cleaved oocytes (Table 6). However, acrosome and membrane integrities were near to significance (p =0.06 and p = 0.09 respectively) although the full model was not significant (F3,6 = 5.15 p = 0.16). We then conducted a reduced model with the near variables to significance (Table 7). This reduced model was significant (F2,6 = 10.06, p = 0.04). Also, acrosome and membrane integrities were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Relationship between the percentage of cleaved oocytes and sperm parameters was negative for acrosome integrity and positive for membrane integrity (Table 7).
| Independent variable | Coefficient | Standard error | t | p |
|---|---|---|---|---|
| Constant | 0.928 | 0.177 | 5.246 | 0.013 |
| NAR | −1.034 | 0.238 | −4.340 | 0.023 |
| HOST | 0.654 | 0.190 | 3.452 | 0.041 |
- Total model R2 = 0.870. NAR, intact acrosome; HOST, membrane integrity.
Discussion
The interest in preserving germplasm of the wild species has resulted in a greater attention to the possible recovery and cryopreservation of the sperm from epididymides of dead animals. Such is the situation of many cervid species like Iberian red deer, in which killed males may represent a readily accessible source of sperm for captive breeding programmes. However, up to now, very little information is available on the relationship between the characteristics of the Iberian red deer cryopreserved epididymal spermatozoa and the potential ability of these spermatozoa to fertilize oocytes in vitro. Thus, in the present study we determined the relationship between some sperm parameters assessed after thawing in an Iberian red deer and the percentage of cleaved oocytes recovered from sheep.
Cryopreservartion causes sperm damage, decreased sperm viability and funcionality as also lower fertility (Watson 2000; Watson and Fuller 2001). Therefore, before artificial insemination with thawed sperm, it is essential to assess the quality of sperm samples. In vitro fertilization offers the best method for assessing sperm function during fertilization. However, these analyses are expensive and time-consuming. An alternative is to find sperm parameters related to the in vitro fertility to know the fertility of thawed sperm samples.
The use of heterologous IVF techniques to evaluate sperm quality is an alternative to homologous IVF as the recovery of oocytes in wild animals is difficult and it is often not possible to obtain a high number of the same ones. However, some researchers have used different techniques to the one carried out in our study. Thus, the hamster oocyte penetration test has been used successfully to assess the sperm fertilizing ability of the bull, the stallion and the red deer (Brackett et al. 1982; Brahmkshtri et al. 1999; Soler and Garde 2003). In this study, we used heterologous IVF with zona-intact matured sheep oocytes derived from slaughterhouse ovaries, because we can obtain a high number of the same ones and avoid killing a large number of animals for experimental purposes. Furthermore, this system requires less maintenance than laboratory animal oocytes. Other researchers, similarly, have used zona-intact matured bovine oocytes to assess spermatozoa of African Antelope with satisfactory results (Roth et al. 1998, 1999; Kouba et al. 2001).
We showed that Iberian red deer thawed epididymal spermatozoa fertilize zona-intact matured sheep oocytes. Nevertheless, the mean value of cleaved oocytes was low (21.90%). This could be due to the low reproductive status of the sheep used in this study. These animals are usually old sheep of little value in Spain. In recent experiments carried out by our group using a homologous IVF test with sheep oocytes of similar quality we obtained a low percentage of cleaved oocytes, being higher when the oocytes were recovered from ovaries of young calves (data not shown). Nonetheless, some males fertilized until 55% of sheep oocytes in some replicates. Likewise, Comizzoli et al. (2001) found results similar to ours, using zona-free matured bovines oocytes and red deer epididymal sperm. On the other hand, as in other studies with other species a great variability was observed between the replicates of IVF (O'Meara et al. 2005). This variability could be due to the different age, the body condition and the handling of the sheep.
Recent studies stated that the sperm characteristics of the recently thawed samples were not good indicators of the success of the IVF in thawed sperm samples of oryx (Roth et al. 1999). These authors have found a positive correlation among the longevity of the motility of the sperm samples incubated after the thawing and IVF. Therefore, in our study seminal parameters were measured immediately after thawing and after 2 h of incubation.
Some sperm parameters were related with the IVF. Thus, acrosome and membrane integrities assessed after incubation of thawed samples at 37°C for 2 h were related to the percentage of cleaved oocytes. The relationship between acrosome integrity and in vitro fertility was negative. Our results did not agree with those of Gadea and Matás (2000) and Tartaglione and Ritta (2004) in whose studies the relationship found was positive. The differences between previous findings and ours could be due to differences in the source of the spermatozoa. It is well known that there are important differences in the physiological characteristics of epididymal vs ejaculated spermatozoa, specially in their membranes properties (Rath and Niemann 1997). Furthermore, epididymal spermatozoa have not been exposed to the complex secretions of the accessory sex glands; these secretions alter both the chilling sensitivity and the acrosome membrane resistance. Perhaps in these samples and in this species acrosome-reacted spermatozoa is needed to fertilize the zona-intact sheep oocytes. Differences in the acrosomal status to initiate binding to the zona pellucida have been shown between species in several studies (Huang et al. 1981; Kusan et al. 1984; Myles et al. 1987; Fazeli et al. 1997). Thus, in our study the males with a less responsive acrosome react after incubation at 37°C for 2 h in a non-capacitating medium and are less likely to fertilize sheep oocytes. In a similar previous study carried out by us with Iberian red deer epididymal sperm, a positive relationship was found between acrosome integrity and percentage of penetrated eggs (Soler and Garde 2003). These differences with this work can be due to differences in the treatment of the sperm. Thus, we used cryopreserved sperm and in the other study the sperm used was fresh. Also, the oocytes source was different (hamster vs sheep in the previous work) and in this study we used zona-intact oocytes. All these differences can determine a different acrosome status to carry out the fertilization.
The other sperm parameter related to the IVF was membrane integrity. Thus, the relationship between membrane integrity assessed by HOST after incubation and the percentage of cleaved oocytes was positive. Similar results have been shown by Van der Ven et al. (1986); Soler and Garde (2003) and Tartaglione and Ritta (2004) in men, red deer and bull respectively. Therefore, the membrane integrity assessed by this method is a good indicator of in vitro fertility.
On the other hand, the SM assessed after incubation was not related to the percentage of cleaved oocytes. Our results are different from the study carried out by Roth et al. (1999) and Soler and Garde (2003) in antelope and red deer respectively. In those studies relationships between a motility index and percentage of cleaved oocytes and penetration eggs were found. Nevertheless, in both cases the SM was assessed together with movement quality and perhaps this index is more related to the in vitro fertility than if we simply evaluate the SM as in this study.
Conclusion
Cryopreserved epididymal sperm from Iberian red deer was capable of fertilizing zona-intact sheep oocytes. However, the low mean value of cleaved oocytes and it could be due to the oocyte quality. After an incubation of sperm samples at 37°C for 2 h some sperm factors were related to the IVF. Thus, acrosome integrity was related negatively to the percentage of cleaved oocytes, being this relationship positive for the membrane integrity assessed by HOST.
Acknowledgements
The authors thank Murgaca for their collaboration in the collection of the samples used in this work. Ana J. Soler enjoyed a studentship from the MEC.
