Single-Layer centrifugation has been used to improve the quality of sperm samples in several species. However, where stallion or boar semen is to be used for AI, larger volumes of semen have to be processed than for other species, thus limiting the effectiveness of the original technique. The objective of the present study was to scale up the SLC method for both stallion and boar semen. Stallion semen could be processed in 100 mL glass tubes without a loss of sperm quality, and similarly, boar semen could be processed in 200 mL and 500 mL tubes without losing sperm quality. The results of these preliminary studies are encouraging, and larger trials are underway to evaluate using these methods in the field.
Single Layer centrifugation (SLC) through a colloid has been advocated as a biomimetic method for improving sperm quality in artificial insemination (AI) doses, since it selects morphologically normal spermatozoa with intact membranes and good chromatin integrity in a similar manner to the selection processes occurring in the female reproductive tract [
A major obstacle to the use of SLC to process stallion or boar spermatozoa for (AI) is the large volume of the ejaculate and the relatively high sperm number in the insemination dose [
SLC-4.5. The tube on the left shows the SLC-4.5 prior to centrifugation, with 4.5 mL extended boar semen on top of 4.0 mL Androcoll-P. The tube on the right shows the preparation after centrifugation with the sperm pellet clearly visible in the bottom of the tube. Note the white line marking the interface between semen and colloid after centrifugation. This consists of spermatozoa that have not been able to pass into the colloid because of poor motility, abnormal morphology, or damaged chromatin.
SLC-15. The various steps in the preparation of SLC-15 with stallion semen. (a) The first 1.5 mL (the most sensitive) is added from a Pasteur pipette to avoid disrupting the colloid surface and create a sharp interface; (b) The remaining semen is added from a disposable pipette; (c) All the semen has been layered on the colloid (note that the interface between semen and colloid is clearly delineated); (d) After centrifugation, the supernatant is removed by aspiration and the sperm pellet is aspirated using a clean pipette.
The purpose of the preliminary studies reported here was to investigate the possibility of scaling-up SLC with species-specific formulations of Androcoll (Androcoll-E or Androcoll-P, where the suffix denotes equine or porcine) to process larger volumes of stallion and boar semen than hitherto attempted. The different scale-up sizes are referred to henceforth by the volume of extended semen processed; for example, SLC-15 refers to processing 15 mL extended semen.
Warmblood stallions (4) of breeding age (7 to 23 years old) and known fertility were housed under standard husbandry conditions at a commercial stud in Sweden. Semen was collected during the month preceding the breeding season at the Division of Reproduction, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden, as part of a teaching demonstration. The stallions were allowed to mount a phantom and ejaculated into a warmed artificial vagina (Colorado type), the semen being collected into a warm plastic bottle with an in-line filter to capture gel. The semen was immediately extended 1 : 1 with warm (35°C) INRA96 semen extender (IMV Technologies, l’Aigle, France). After measuring the sperm concentration with the Nucleocounter SP-100 [
Four boars aged 3-4 years, (Swedish Landrace, Swedish Yorkshire and two Norwegian Landrace) were housed at the Division of Reproduction, Swedish University of Agricultural Sciences, Uppsala, Sweden, under standard husbandry conditions [
Aliquots (150 mL) of boar semen from one of the boars used for experiment 2, extended to 100 × 106 spermatozoa/mL, were prepared by (i) SLC on top of 150 mL Androcoll-P (Large) in a 500-mL tube (SLC-150) and (ii) by simple centrifugation (sperm “washing”). After centrifuging at 300 g for 20 min, the supernatant was removed, and the sperm pellets were resuspended in Beltsville Thawing Solution (BTS) with added bovine serum albumin (5 mg/mL). In another experiment, aliquots of semen were prepared by SLC-150 or by SLC-15 (150 Androcoll-P (S) and 15 mL Androcoll-P Large, resp.) (Figure
SLC-150. Notes: (a) After centrifugation, the different layers can be seen, comprising 150 mL seminal plasma and extender on top of 150 mL colloid, with the sperm pellet in the bottom of the tube; (b) Removing the supernatant after centrifugation using a water pump; (c) Aspirating the sperm pellet.
A summary of the volumes of colloid and semen and the proposed nomenclature for the various techniques is given in Table
Summary of the volumes of colloid and extended semen*, centrifuge tubes, and centrifuges used in these experiments (modified from [
Species | Tube size | Volume of colloid (mL) | Volume of extended semen* (mL) | Name of technique | Centrifuge |
---|---|---|---|---|---|
Stallion/boar | 12 mL | 4.0 | 4.5 | SLC-4.5 | 1 |
Stallion/boar | 50 mL | 15 | 15–18 | SLC-15 | 1 |
Stallion/boar | 100 mL | 20 | 25 | SLC-25 | 2 |
Stallion/boar | 200 mL | 60 | 60 | SLC-60 | 3 |
Boar** | 500 mL | 150 | 150 | SLC-150 | 1 |
*Extended to give a concentration of approximately 100 × 106 spermatozoa/mL.
**this technique has not been attempted yet with stallion semen but is theoretically possible.
Centrifuge 1: for example: Hereaus Multifuge, Kendro, Osterode, Germany;
Centrifuge 2: for example: Sigma Laboratory Centrifuge, Osterode, Germany;
Centrifuge 3: for example: Centra MP 4R, International Equipment Company, MA, USA.
Progressive motility (PM) in the different samples is shown in Figure
Progressive motility in uncentrifuged, SLC-4.5, and SLC-25 sperm samples (
Mean values (±SD) for PM, normal morphology, and membrane integrity of the boar sperm samples are shown in Table
Mean values (±SD) of parameters of sperm quality in SLC-selected (SLC-15 and SLC-60) boar sperm samples (
Sample | Progressive motility (%) | Normal morphology (%) | Membrane integrity (%) |
---|---|---|---|
SLC-15 | 87 ± 11 | 94 ± 5 | 94 ± 3 |
SLC-60 | 87 ± 11 | 87 ± 3 | 95 ± 3 |
Note: SLC-15 and SLC-60 refer to 15 mL extended semen on top of 15 mL Androcoll-P (Large) and 60 mL extended semen on top of 60 mL Androcoll-P, respectively.
Sperm motility in the sperm samples was as follows: immediately SLC-150 82%, wash 61%, and control 68% and after storage for 7 days at 16°C–18°C (SLC 75%, wash 55%, and control 70%). The yield from SLC-150 was 80%–86%. The patterns of sperm motility are shown in Figure
Sperm tracks obtained during motility analysis of uncentrifuged and SLC-selected boar sperm samples using the SpermVision motility analyzer. Note: red dots indicate immotile spermatozoa, green tracks indicate progressively motile spermatozoa, and yellow tracks indicate locally motile spermatozoa.
Uncentrifuged sperm sample
SLC-selected sperm sample from the same boar
The purpose of these preliminary experiments was to investigate whether the SLC-technique could be scaled up to facilitate processing larger volumes of extended stallion and boar semen than hitherto routinely used. The volumes of Androcoll chosen gave approximately the same height of the colloid column in the different centrifuge tubes, since our previous work had suggested that this is an important factor in the success of the technique [
In the first experiment, parameters of stallion sperm quality were comparable in the large-scale SLC-samples and smaller volume SLC, suggesting that it is possible to scale up the technique for stallion semen in 100 mL glass tubes without compromising semen quality. These results are in agreement with previous results, which showed that SLC-25 (in 100 mL tubes) was possible for boar semen [
The preliminary results of the boar semen experiments reported here show that scaling up the technique for use in 200 mL and 500 mL tubes is possible. The results resemble those obtained previously for SLC-60 (in 200 mL tubes) with stallion semen [
SLC-60 selected spermatozoa were used successfully for freezing, showing higher postthaw progressive motility than SLC-15. This result may be due to a higher sperm concentration in the sperm pellet. If this is the case, it should also be possible to improve the freezing of SLC-15 selected spermatozoa by centrifuging the sperm suspension one more time to concentrate the sperm pellet before resuspension in the cryoprotectant. Alternatively, use of the SLC-150 would allow all of the extended sperm-rich fraction (SRF) of the boar ejaculate to be processed in three or four 500 mL tubes in one centrifugation cycle. This method could be of interest for the boar semen industry, and a larger trial is currently underway to assess the usefulness of the method.
The SLC technique is more convenient and less time consuming than density gradient centrifugation (DGC), which has previously been used occasionally to prepare animal semen [
In conclusion, the SLC technique can be scaled up to process 25 mL stallion semen or 60 mL and 150 mL boar semen without loss of sperm quality.
The authors give thanks to the animal husbandry staff at the Division of Reproduction, SLU, for looking after the animals, Lennart Söderquist for arranging the stallion semen samples, Patrice Humblot for the statistical analysis, and Annika Rikberg and Karin Selin-Wretling for the morphological evaluation. This work funded by the Swedish Foundation for Equine Research, the Swedish Farmers’ Foundation, the C.F. Lundström Foundation and Jordbruksverket, Sweden.