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From the Departments of * Biochemistry and
Molecular and Cell Biology and Animal
Sciences, School of Veterinary Medicine, University of Zaragoza, Miguel
Servet, Zaragoza, Spain.
| Correspondence to: Dr Teresa Muiño Blanco, Departmento de Bioquímica y Biología Molecular y Celular Facultad de Veterinaria, Miguel Servet, 177, 50013-Zaragoza, Spain (e-mail: muino{at}posta.unizar.es). |
| Received for publication March 2, 2004; accepted for publication June 15, 2004. |
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Abstract |
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Key words: Sperm quality, reproductive efficiency, IUI
At most artificial insemination (AI) centers, the conventional principal laboratory test for the assessment of semen quality is an estimation of the percentage of motile cells and the vigor of that motility. That method, which is an indirect method of assessing metabolic activity, shows an inherent variability in results because it is influenced by subjective counting errors (Graham et al, 1980). Yet, most commercial semen-processing operations routinely use that measure as an initial estimate of semen quality. Repeatedly higher motility tests can be obtained by computer-aided sperm analyzers (Davis and Siemers, 1995). By this approach, certain specific motility patterns have been related to fertility of frozen-thawed semen (Budworth et al, 1988; Zhang et al, 1998).
The most important mechanisms involved in fertilization, including capacitation, acrosome reaction, and binding of spermatozoa to the egg surface are believed to depend on the functionality and integrity of the sperm membrane. For that reason, numerous techniques have been developed to provide a more specific evaluation of membrane functionality. Those techniques include membrane integrity assessment with the use of different fluorochromes (Garner et al, 1986; Harrison and Vickers, 1990; De Leeuw et al, 1991; Valcárcel et al, 1994), the hypo-osmotic swelling test (Jeyendran et al, 1984; Correa and Zavos, 1994), assessment of mitochondrial activity (Garner et al, 1997; Windsor, 1997; Thomas et al, 1998; Nagy et al, 2003), evaluation of acrosomal status with the use of fluorescent lectins (Mortimer et al, 1987; Cross and Meizel, 1989) or antibodies (Fenichel et al, 1989), and the zona-free hamster ovum test (Yanagimachi, 1984).
Mitochondrial activity is most often evaluated with rhodamine 123 (Rh 123), a cationic compound that accumulates in the mitochondria as a function of dye concentration and sperm number (Windsor and White, 1993) and transmembrane potential (Chen, 1988; Al-Rubeai et al, 1993). Simultaneous assessment of both viability and mitochondrial activity, with the use of stains such as ethidium bromide or propidium iodide (Evenson et al, 1982) along with Rh 123, can be used to test storage and sperm survival (Magistrini et al, 1997).
Despite the utility of standard semen parameters in evaluating seminal quality, most single parameters (taken into account individually) are weakly correlated with in vivo fertility. In the last years, numerous studies have questioned the validity of these simple tests as predictors of fertility (Brahmkshtri et al, 1999; Larsson and Rodriguez-Martinez, 2000; Foote, 2003; Rodriguez-Martinez, 2003). Fertility traits are classified either as compensable or uncompensable (Saacke et al, 2000). Compensable traits include defects, such as those associated with sperm viability or morphology, that can be compensated for insemination with a large number of spermatozoa. If an animal has defects in compensable traits, reduced fertility occurs when a suboptimal number of spermatozoa are inseminated. When excess spermatozoa are inseminated, only the traits that are considered uncompensable, such as chromatin aberrations, affect fertility. Likewise, when comparing the fertilizing ability of semen samples, a relatively small number of spermatozoa per insemination should be used so as not to obscure possible differences in fertility (Pace et al, 1981; Shannon and Vishwanath, 1995). Furthermore, to maximize reproductive efficiency, a reduction in the number of spermatozoa used per insemination is an important objective in animal production.
AI plays an important role in sheep breeding but is limited by the relatively poor fertility achieved with stored semen. The success of this procedure in sheep is limited by the short length of time that ram sperm can be stored as a liquid. Intrauterine insemination (IUI) has improved fertility rates. IUI techniques used with low sperm concentrations have been used with cow (Seidel et al, 1997), horse (Buchanan et al, 1999), sheep (Ham et al, 2000), and pig (Krueger et al, 1999; Krueger and Rath, 2000; Martinez et al, 2002; Roca et al, 2003) with acceptable fertility results. In any case, a prerequisite for a successful AI system in sheep is a diluent that maintains sperm motility and viability during cooling to subambient temperature (usually 15°C). Sperm diluents also are crucial in avoiding loss of viability from the dilution and washing of sperm cells, commonly done to remove seminal plasma or to achieve a high sperm concentration. Various procedures for the separation of spermatozoa have been applied to fresh ejaculates to rid the spermatozoa of seminal plasma and other contaminants (eg, dead and abnormal spermatozoa that alter motility, viability, or morphology of sperm cells; Shannon and Curson, 1972; Lindemann et al, 1982) that reduce fertility (Saacke et al, 1994). Earlier (García-López et al, 1996), we reported the development of a dextran/swim-up procedure for the selection of high-quality ram spermatozoa in a medium containing CaCl2 and NaCO3H. In this study, we present a comparative analysis of the sperm quality produced by the dextran/swim-up procedure when used with or without capacitating compounds (CaCl2 and NaCO3H). In addition, we compared sperm survival in both selected samples and in the raw semen following incubation at 2 temperatures: 15°C (the typical temperature at which ram semen is stored until artificial insemination) and 30°C (the mean temperature at which semen is processed in our laboratory). Finally, we determined whether differences found in sperm quality were reflected in differences in fertilization. For this purpose, superovulated ewes were inseminated via the intrauterine route with the use of a low number of spermatozoa per insemination to enhance possible differences in fertilization rate.
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Materials and Methods |
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Semen Samples
Two sperm selected samples were obtained by a dextran/swim-up procedure
(García-López et al,
1996) performed with SM medium as SM+ (200 mM sucrose, 50 mM NaCl,
18.6 mM sodium lactate, 21 mM HEPES, 10 mM KCl, 4 mM NaHCO3, 2.7 mM
CaCl2, 2.8 mM glucose, 0.4 mM MgSO4, 0.3 mM sodium
pyruvate, 0.3 mM K2HPO4, 1.5 IU/mL penicillin, and 1.5
µg/mL streptomycin, pH 6.5) or SM- (devoid of CaCl2 and
NaHCO3). The selected samples (SS) were called SS+ and SS-,
respectively. The number of recovered cells in each selected sample was
approximately 3 x 108 cells/mL in 2.25 mL of SM medium
(approximately 50% of sperm recovery). With each of these samples, 3
insemination doses were prepared. Control samples consisted of diluted raw
semen in SM medium. In all experiments, dilution was adjusted to obtain the
same concentration as in selected samples.
For intrauterine inseminations, sperm samples of 5–10 x 107 cells were packaged in 0.25-mL straws and kept at 30°C throughout the experiment.
Assessment of Semen Parameters
In this study, the sperm quality parameters assessed included sperm
concentration, progressive individual motility, hypo-osmotic swelling, cell
viability, and mitochondrial activity. Those parameters were measured
immediately after semen dilution at room temperature (control sample) and
after the swim-up procedure (SS+, SS-). To evaluate sperm survival, each
sample was divided into 2 aliquots, one was maintained at 30°C in a heater
and the other was maintained at 15°C in an ice water bath over incubation
periods of 2, 4, and 6 hours.
Sperm concentration was calculated in duplicate with a Neubauer chamber (Marienfeld, Lauda-Königshofen, Germany).
Sperm motility was subjectively assessed by visual estimation with a television microscopy system (100x) maintained at 37°C (Evans and Maxwell, 1987). The percentage of progressively motile spermatozoa was estimated at 5% increments, and the same person performed the evaluation throughout the study.
The hypo-osmotic swelling (HOS) test was used to evaluate the functional activity of the sperm membrane. The procedure was described by Jeyendran et al (1992) and adapted for ram semen by García-Artiga (1994). The assay was performed by mixing 10 µL of the sample with 1 mL of a hypo-osmotic solution (7.35 g sodium citrate·2H2O and 13.51 g fructose in 1 L of distilled H2O, adjusted to 100 mOsm/L) and incubating at 37°C for 30 minutes. After treatment, sperm exhibited several of the characteristic responses that are induced by intracellular swelling, but only those sperm having a curling tail were considered to be HOS test–positive. Two hundred cells were evaluated by counting in at least 5 fields under a phase contrast microscope at 400x magnification.
Cell viability is defined here as both intact plasma and acrosomal membranes. It was assessed (Harrison and Vickers, 1990) by fluorescent staining with carboxifluorescein diacetate (CFDA) and propidium iodide (PI; Sigma Chemical Co, St Louis, Mo). The cells were then examined under a Nikon fluorescence microscope with a B-2A filter, and the number of fluorescein-positive (membrane-intact) spermatozoa and PI-positive (membrane-damaged) spermatozoa per 100 cells were estimated and recorded. Nonviable cells can be classified into 2 groups: with damaged acrosome or with intact acrosome. The last group represents a small sperm subpopulation doubly stained with both CFDA and PI. At least 200 cells were counted in duplicates for each sample.
Mitochondrial activity was evaluated with the use of a specific probe that included staining with Rh 123 coupled with PI stain to discriminate between living and dead spermatozoa (Evenson et al, 1982). An aliquot of 500 µL of diluted sample containing 6 x 106 cells/mL was mixed with 5 µL of formaldehyde (1.7 µM), 5 µL of PI (7.3 µM), and 5 µL of Rh 123 (0.2 mM) and incubated at 37°C for 30 minutes in the dark. Cells were examined under a Nikon fluorescence microscope, and 4 subpopulations of cells were observed: cells with functional mitochondria with (Rh+/PI-) or without (Rh+/PI+) an intact plasma membrane, and cells without functional mitochondria with an intact (Rh-/PI) or damaged (Rh-/PI+) plasma membrane.
Hormonal Treatment and IUI
A total of 56 Rasa Aragonesa ewes from several farms belonging to ANGRA
were kept at the Veterinary School under uniform nutritional conditions. In
each experiment, 9 ewes were inseminated to compare 3 different samples
(control and selected samples obtained from the same semen pool and 3 ewes) in
each of the 3 experiments. The study was blind because the intrauterine
inseminations and embryo recovery were performed mechanically.
Estrus was synchronized by intravaginal sponges containing 30 mg fluorogestone acetate (Chrono-gest; Intervet, Salamanca, Spain) inserted for 14 days. Ewes were superovulated with a total dose of 176 NIH-FSH-S1 units of NIADDK-oFSH-17 (Ovagen ICP-LTD Ltd, Auckland, New Zealand) in 8 doses administered IM at 12-hour intervals and beginning 72 hours before intravaginal sponge removal. Each animal received the dose in 10 mL of solution subdivided into 2 x 2-mL, followed by 6 x 1-mL, injections. Ewes were checked for estrus every 8 hours.
Ewes were inseminated by laparoscopy with the use of 0.125 mL of diluted semen (2.5, 3.5, or 5 x 107 sperm, depending on the experiment) per uterine horn 64 or 52 hours after progestagen removal, the intervals recommended by the Food and Agriculture Organization of the United Nations (FAO) for normal and superovulated ewes, respectively (Baril et al, 1993). Embryos were collected 6 days after insemination by midventral laparotomy. Ewes were anesthetized by IM injection with 0,4 mL of 2% xylazine. Five minutes later, 10 mL of sodium thiopental (20 mg/mL, Thiobarbital; Braun Medical, Jaen, Spain) was administered IV. Both uterine horns were exposed and flushed with prewarmed phosphate-buffered saline supplemented with 1% bovine serum albumin (Sigma) and antibiotics (penicillin and streptomycin). To minimize the development of postoperative abdominal adhesions, the reproductive tract was flushed with a 2.5% heparin solution in saline before closure.
Experimental Design and Presentation of Data
Three samples (control, SS+, and SS-) were compared by inseminating 9 ewes
in each experiment (3 ewes with each type of sample per experiment).
Experiments with both protocols (52 and 64 hours) were carried out
independently.
Fertilization rate was measured as the percentage of embryos recovered from the uterine horns 6 days after insemination, relative to the total number of corpora lutea counted in the ovaries. The number of corpora lutea in the ovaries indicates the number of ovulations produced, and the recovered embryos and oocytes correspond to the number of fertilization successes and failures, respectively.
Statistical Analysis
Results are presented as means ± SEM of the number of samples
included in the analysis. In the study of seminal quality, means were compared
by analysis of variance tests to determine whether there were significant
differences between samples and between conditions of storage. In the
insemination experiments, correlations between quality parameters and
fertilization were determined by the Pearson coefficient with the use of SPSS
software (version 11.5).
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Results |
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Sperm viability (CFDA/PI staining) and mitochondrial functionality (Rh+/PI-) were significantly positively correlated (r = .76, P < .001).
Maintenance of Sperm Quality
To determine whether the presence (SS+) or absence (SS-) of capacitating
compounds affected sperm survival, a comparative study of the preservation of
sperm quality in control and selected samples was performed. Because of the
highly significant correlation between viability and mitochondrial
functionality, in these experiments, only CFDA/PI staining was used to assess
sperm membrane integrity (Harrison and
Vickers, 1990).
To determine whether temperature affects sperm quality during in vitro storage, samples were incubated at 15°C or 30°C for 6 hours, and standard semen parameters were recorded every 2 hours. Motility was better maintained at 15°C (Figure 2a) than at 30°C (Figure 3a), although a progressive decrease in the percentage of motile sperm was observed at both temperatures in all samples over time. Selected samples still maintained a motility value of 46% after 6 hours of incubation at 15°C, whereas only 27% of sperm were motile in control samples (P < .001). No significant differences were found between SS+ and SS-, suggesting that the presence or absence of capacitating compounds in the selection medium did not influence the maintenance of motility. Statistical analysis confirmed the convenience of maintaining the swim-up samples at 15°C rather than at 30°C (P < .001 and P < .0001 for SS+ and SS-, respectively, after 6 hours of incubation).
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In the selected samples, sperm viability was well preserved at 15°C throughout the total incubation period (6 hours) and was significantly higher than in control samples (Figure 2b; P < .0001). Moreover, swim-up samples had significantly higher viability than those of the controls (P < .0001) after 2 hours of incubation at 30°C, even though control samples preserved higher viability values at 30°C than at 15°C (P < .01). Adding CaCl2 and NaHCO3 to the selection medium did not affect sperm viability because similar results were obtained with SS+ and SS-.
The response to the HOS test had a drastic decrease after 2 hours of incubation at 15°C, then remained constant until the end of the 6-hour period without significant differences between samples (Figure 2c). At 30°C, although selected samples showed a more progressive decrease than control samples at 2 (P < .01) and 4 hours (P < .0001) of incubation (Figure 3c), there were no significant differences at the end of the 6-hour incubation period (Figure 3c). Again, similar values were found for both selected samples.
After 2 hours of incubation, swim-up samples showed significantly higher quality at 15°C and 30°C compared with controls, and the significance of the differences was even greater at 30°C than at 15°C. After a long incubation period at 15°C, motility and viability were significantly better preserved in selected than in control samples. At 30°C, however, the differences between samples after 2 hours of incubation disappeared when incubation was extended to 6 hours.
Evaluation of Fertilization Rate
Given the differences between control and swim-up–selected samples in
sperm quality and survival, the second objective of this study was to
determine whether these differences are reflected in the fertilization success
of the samples. Thus, raw semen diluted in SM medium and selected samples (SS+
and SS-) were used for intrauterine insemination in superovulated ewes.
IUI was designed to occur within 2 hours of semen extraction; therefore, we chose 30°C as the storage temperature on the basis of the results of the first part of this study. Fifty-six inseminations were performed: 20 with control samples, 18 with SS+, and 18 with SS-.
Table 2 shows the results of both insemination protocols at intervals of 52 and 64 hours between progestagen removal and AI. The number of corpora lutea seen in the ovaries indicates the number of produced ovulations, whereas recovered embryos correspond to the number of fertilization success (see the "Materials and Methods" section). We established a strict criterion of fertilization rate because only the percentage of recovered embryos was considered, not the percentage of recovered oocytes and embryos.
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Obtained fertilization values were much higher in SS- than in SS+ or control samples (Table 2). When SS- was used for insemination, the fertility rate was 67.7% and 53.2% when the intervals between progestagen removal and AI were 52 and 64 hours, respectively. Control samples failed in their ability to fertilize following the protocol recommended by FAO (Baril et al, 1993) for superovulated ewes (52 hours), with only 1 embryo recovered (Table 2). Similarly, fertilization achieved with SS+ was also extremely low (7 embryos). Results were substantially better with the 64-hour protocol, with fertilization rates of approximately 40% observed in both samples.
Comparative analysis of mean fertilization values obtained with all samples revealed significant differences between both protocols for control (P < .001) and SS+ (P < .01). The fertilization rate obtained with SS-, however, was not affected by the protocol used (Table 3). With the 52-hour protocol, fertilization values for SS- were significantly (P < .001) higher than those of control and SS+. It is noteworthy that those differences were not due to variations in the ovulation rate because no significant differences in the number of corpora lutea were observed between samples or protocols.
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To determine whether the fertilizing ability of the semen samples was influenced by the inseminated sperm concentration, we carried out a comparative analysis of the results obtained with the 3 types of samples inseminated following the 64-hour protocol because the type of sample did not affect the results under that protocol. Sperm samples were placed in 1 of 3 categories depending on the cell concentration per insemination dose: low concentration (50 million), medium concentration (50–70 million), and high concentration (70–100 million). Comparing the 3 sperm concentrations and all samples, no statistical differences were found, although the highest embryo yield was obtained with the highest concentration (Table 4). Interestingly, at low sperm concentrations, SS- produced the best fertilization rates (43% compared with 28% and 33% for control and SS+, respectively). No significant differences were observed when comparing the 3 samples with each concentration, probably because of variability of results and the discrete nature of the data.
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Despite the differences between control and selected samples in sperm quality, no significant correlations were found between the standard analyzed parameters and the fertilization rate achieved with the 3 types of samples in this study, which agrees with our previous results on ram field fertility by cervical insemination (Perez-Pe et al, 2002).
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Discussion |
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Most selection procedures are designed to increase the proportion of motile sperm and reduce the percentage of damaged spermatozoa. Often, the success of a sperm preparation method is measured by assessing the percentage of motile spermatozoa recovered (Alvarez et al, 1993). It has been reported, however, that some spermatozoa showing good motility have membrane damage (Valcárcel et al, 1994). For that reason, most studies also include an evaluation of the structural and functional integrity of sperm membranes as an indicator of the efficacy of a selection procedure (Correa et al, 1997; Martí et al, 2003). In this study, we used mitochondrial activity along with motility, viability, and the HOS test as sperm quality parameters. We demonstrated the value of the dextran/swim-up procedure in the selection of high-quality sperm in media both with and without capacitating compounds. However, no significant differences were found between the selected samples, which suggests that the addition of CaCl2 and NaCO3H to the selection medium did not influence sperm quality.
Not only was sperm quality of selected samples higher than in the control samples, but it also was better preserved following incubation at 15°C. That is important because 15°C is the temperature usually used to store Rasa Aragonesa semen in AI centers; therefore, an important prerequisite for a successful AI system is the maintenance of semen quality parameters during storage at this temperature.
When samples were incubated at 30°C, differences in quality parameters between control and selected samples were absent after 6 hours of incubation, possibly because of better preservation of sperm quality in control samples. This result is consistent with studies that showed that sperm viability in raw semen is better maintained at temperatures higher than 15°C in the first 4–8 hours, although after 8 hours, viability drops sharply (Huo et al, 2002). It is worth noting that during shorter incubation periods (2 hours), significant differences were observed between raw semen and swim-up samples in all of the parameters analyzed following incubation at 30°C, with some of the differences much more significant than at 15°C. For that reason, we chose 30°C as the temperature for maintaining samples until IUI. Our results showed that differences in sperm quality parameters were clearly evident when insemination was performed under limiting conditions. Selected samples without capacitating compounds (SS-) had higher fertilizing ability when ovulation and insemination were possibly not fully synchronized, as appeared to occur in the 52-hour protocol, or when sperm concentration was very low. The low fertilization rate achieved with SS+ in the 52-hour protocol might be because of the delay in ovulation relative to the time of insemination (when insemination could be anticipated to the ovulation) because all other variables in the study were unmodified. Whether or not the majority of sperm in these samples are capacitated, they might not be able to survive the delay in the female tract.
The use of ram sperm samples selected with the dextran/swim-up procedure without capacitating compounds could be a suitable choice for intrauterine insemination and could be helpful in cases in which ovulation and insemination times are not well coordinated.
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Acknowledgments |
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Footnotes |
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), hypo-osmotic swelling (HOS)
score (
), viability (
), and mitochondrial functionality (
)
in selected samples with respect to control samples. Results are expressed as
means ± SEM (n = 19).
