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Evaluation of Frozen-Thawed Semen
T Katila, E Koskinen* and M Andersson
Department of Clinical Veterinary Sciences, Saari Unit, Finland Saarentaus
*Department of Animal Science, Finland, Helsingin Yliopisto
University of Helsinki

Introduction

There is considerable variation between individual stallions in how well their semen retains its fertilizing capacity after freezing and thawing. It has been estimated that only 20% of fertile stallions produce sperm that survive the freezing and thawing processes (Tischner 1979). Although our knowledge and techniques have improved within the last 20 years, a considerable proportion of stallions are still not suitable for semen freezing. Development of freezing methods requires in vitro tests that correlate with in vivo fertility, but controlled breeding trials with horses are expensive (Loomis 1999). The slow progress in the development of freezing techniques is partly explained by the lack of reliable laboratory methods. Some in vitro methods work reasonably well in the assessment of fresh but not frozen semen, the best example being motility evaluation. In spite of its limited applicability, motility is the most commonly used parameter in the evaluation of frozen-thawed semen, in both laboratories and stud farms, because it is easily accessible and quick to perform. A combination of laboratory tests should enable better assessment of the fertility of cryopreserved stallion semen (Blach et al. 1989).

There is very little data about the correlation between semen evaluation tests and pregnancy rates after insemination with frozen semen. Although good correlations have been found between different tests, it does not mean that they would correlate with fertility.  The purpose of this study was to find out which tests used to evaluate stallion semen would correlate with foaling rates of mares inseminated with frozen semen. Our previous study indicated that incubation of the semen sample at +37oC might be useful in the evaluation of frozen-thawed semen (Katila et al. 1999). Therefore, the evaluation tests were performed immediately after thawing and after 3 hours incubation at +37oC.

Materials and Methods

            Frozen semen doses were available from 31 commercially used stallions. The semen had been frozen between the years 1988 and 1998 in 5 different countries: 22 in Sweden, 5 in Finland, 2 in Italy, 1 in Denmark, and 1 in Germany. Semen from 23 stallions had been frozen in 2.5-ml straws and 8 in 0.5-ml straws. Thirteen stallions were American Standardbreds, one stallion was an English thoroughbred, and the remaining 7 were various types of riding horses. Sufficient foaling data – at least 5 mares and 10 cycles – were available from 23 stallions. Foaling data originated from Finland and Sweden from the years 1989 to 1999. The average number of mares/stallion was 35 (range 5 – 121). The average foaling rate was 59% (range 11 – 91%). Seventeen stallions had a foaling rate >50% and 6 had a foaling rate <50%.

Semen evaluation

The following tests were done from 1 to 3 straws on one day immediately after thawing and after 3-hour incubation at +37oC extended in Kenney’s skim milk extender (Kenney et al. 1975): 1) resazurin reduction test by a fluorometer, 2) plasma membrane integrity by CFDA/PI (carboxyluoresceindiacetate/propidium iodide) staining and counting of cells in a fluorescence microscope, and 3) plasma membrane integrity by PI staining and by an automatic fluorometer. In addition, 4) concentration was determined in a Bürker counting chamber. On another day the following tests were done immediately after thawing and after 3-hour incubation at +37oC extended in Kenney’s skim milk extender: 5) motility by a light microscope and 6) motility parameters by a Hamilton Thorn Motility Analyzer, 7) HOST (hypo-osmotic swelling test) by light microscope and 8) HOST by a fluorometer. In addition, concentration was determined in a Bürker counting chamber.

The 0.5-ml straws were thawed for 30 sec in +37oC and the 2.5-ml straws in +50oC for 40 sec. Semen concentration was measured in a Bürker counting chamber, and the total number of spermatozoa/straw calculated. An insemination (AI) dose was one straw when using 2.5-ml straws and from 1 to 10 straws for the 0.5-ml straws. The sample for the longevity test was prepared by extending semen with skim milk extender to a concentration of 40 to 100 x 106 spermatozoa/ml. The sample was kept in a water bath at +37oC for 3 hours.

            For motility evaluation, semen was extended with warm (+30oC) skim milk extender (Kenney et al. 1975) to a concentration of 40 x 106 spermatozoa /ml and incubated at +37oC for 10 min. An aliquot of 7 ml of extended semen was placed on a slide and covered by a cover slip. Motility was evaluated with a light microscope: percentage of progressively motile spermatozoa, percentage of total motility and a velocity score from 1 to 3. Motility was measured also with an automatic sperm analyzer (Hamilton Thorn Motility Analyzer, HTM-S, version 7.2). A 10-ml semen sample was placed onto a Makler chamber, and two chambers were prepared from the same sample. Six fields/chamber were videotaped for 15 sec/field. The videotapes were analyzed for total motility, progressive motility, and path velocity.

For CFDA/PI staining, semen was extended with skim milk extender to a concentration of 50 x 106 spermatozoa/ml. An aliquot of 20 ml of CFDA stock solution consisting of 0.46 mg CFDA in 1 ml of DMSO (dimethylsulphoxide) and 10 ml of PI stock solution (0.5 mg PI in 1 ml of 0.9% NaCl-solution) were taken, mixed with 950 ml of skim milk extended semen and incubated for 8 min at +30oC. A drop of 5 ml was placed on a slide and overlaid with a cover slip (Harrison and Vickers 1990). The proportion of fluorescent cells was counted in 200 cells in a fluorescence microscope (Olympus BH2 with epifluorescence optics) using oil immersion and a fluorescein filter set.

An automatic fluorometer (Fluoroscan Ascent) which reads a 96 well microtitration tray and has an incubation compartment was used for the fluorometric measurement of plasma membrane integrity. The interference filter at the exitation path and that of the emission filter had a maximum transmission at 544 nm and 590 nm, respectively. For the fluorometric assay, 20 mg of PI was dissolved in 1 liter of BTS (Beltsville Thawing Solution) and dispensed in 3 ml aliquots. Equal aliquots (50 ml) of skim milk extended semen sample (40 x 106 spermatozoa/ml) and PI solution were dispensed into the well plate, and the well was shaken gently for 2 min. Spermatozoa from the same samples were killed by unprotected rapid freezing-thawing to have internal control samples consisting of only non-viable cells (100% fluorescence).  The control sample was immersed into liquid nitrogen for 1 min, thereafter it was allowed to stand in a room temperature for 30 sec and then 3 min in a water bath (37oC). Blanks containing 50 ml of diluted extender and 50 ml of PI were analyzed separately for every experiment in 4 replicates. The incubation time was 8 min. The percentage of fluorescence was calculated from the ratio of fluorescence intensities of the rapidly frozen control sample and the sample to be analyzed, taking into account the blank values (Juonala et al. 1999).

 For the resazurin reduction test, 400 mg of resazurin was dissolved in 1 liter of distilled water. One part of this solution and 9 parts of 0.9% NaCl were mixed. An equal volume of this mixture and skim milk extended sperm (40 x 106 spermatozoa/ml) were combined and shaken for 2 min. After that it was incubated for 30 min at 32oC and measured with the fluorometer using the same fluorometer settings as in the plasma membrane viability test (Eriksson et al. 1998).

Hypo-osmotic solution of 100 mOsmol/kg for HOST was prepared by dissolving 4.5 g fructose and 2.717 g sodium citrate in 500 ml of distilled water. An aliquot of 0.5 ml of this solution was mixed with 0.125 ml of semen and incubated at 37oC for 45 min. A 5 ml drop was placed on a slide and overlaid by a cover slip. A total of 200 spermatozoa per sample were evaluated for bent tails in the microscope. For the fluorometric determination of HOST 0.5 ml of the same hypo-osmotic solution (100 mOsmol/kg) was mixed with 0.125 ml of skim milk extended semen (concentration 100 x 106 spermatozoa/ml). The fluorometric method was the same as for PI-stained semen.

Data analysis

Raw correlations were determined by analysis of least squares. The level of significance was set at p<0.05. Only the results of the 23 stallions having sufficient foaling data were included in the statistical analysis.

Results

None of the tests had high correlations with foaling rates. Only HOST performed at 0 (p = 0.03) and 3 hours (p = 0.04) correlated with fertility. When two tests were combined, HOST and CFDA/PI staining at 0 and 3 hours showed significant correlations (p<0.05).

Conclusions

None of the parameters measured correlated well with foaling rates. It seems that tests evaluating plasma membrane integrity (HOST, fluorescent stains) might be more useful in the evaluation of frozen-thawed stallion semen than motility parameters. However, many factors may have influenced the foaling rates. Sperm had been frozen over many years in different laboratories. The number of mares inseminated/stallion was small and inseminated in various conditions over many years. Futhermore, pregnancy rates/cycle would have reflected fertility more accurately than foaling rates, but these figures were not available.

References

1.        Blach EL, Amann RP, Bowen RA, Frantz D 1989. Changes in quality of stallion spermatozoa during cryopreservation: plasma membrane integrity and motion characteristics. Theriogenology 31: 283-298.

2.        Eriksson B, Juonala T, Andersson M, Rodriguez-Martinez H 1998. Viability of frozen-thawed boar semen.  Proceedings of the International Congress on Animal Reproduction,  526.

3.        Harrison RAP, Vickers SE 1990. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. Journal of Reproduction and Fertility 88: 343-352.

4.        Juonala T, Salonen E, Nurttila T, Andersson M 1999. Three fluorescence methods for assessing boar sperm viability. Reproduction of Domestic Animals 34: 83-87.

5.        Katila T, Andersson M 1999. Evaluation of frozen stallion semen. 1st meeting of EEGG, Lopuszna, Poland, 5th-8th September, 15.

6.        Kenney RM, Bergman RV, Cooper WL, Morse GW 1975. Minimal contamination techniques for breeding mares. Proceedings of the Annual Meeting of the American Association of Equine Practioners: 327-336.

7.        Loomis P 1999. Artificial insemination of horses: Where is it going? Proceedings of the Annual Meeting of the Society for Theriogenology, Nashville, Tennessee, 325-336.

8.        Tischner M 1979. Evaluation of deep-frozen semen in stallions. Journal of Reproduction and Fertility, Suppl  27: 53-59.