Table 1. Function and organic osmolyte content of bovine ejaculated spermatozoa processed without cryoprotectants (Protocol C control) and changes relative to the controls, after adding and removing the CPAs listed in Protocols A (multistep) or B (single step).
| CPA | Control (n= 35) | DMSO |
EG |
THP |
GLY |
PD2 |
PD3 |
THE |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Protocol | C | A | B | A | B | A | B | A | B | A | B | A | B | A | B | |
| Mean ± SEM. | ||||||||||||||||
| Mean percentages of internal controls (Protocol C: n = 5) | ||||||||||||||||
| Carnitine (nmol) | per U G6PDH | 18.2 ± 2.3 | 67.1 | 102.1 | 76.8 | 94.5 | 15.4* | 21.5* | 99.8 | 99.8 | 36.4 | 42.2 | 41.8 | 45.4 | 48.4 | 53.8 |
| Glutamate (nmol) | per U G6PDH | 8.1 ± 0.8 | 76.7 | 115.9 | 105.3 | 119.8 | 55.5 | 40.4 | 80.7* | 100 | 49.2* | 58.3* | 90.9* | 98.7 | 92.9 | 94.8 |
| Inositol (nmol) | per U G6PDH | 24.0 ± 1.9 | 101.4 | 100.6 | 102.6 | 81.0 | 69.2 | 80.5 | 119.7 | 99.2 | 76.9 | 102.1 | 101.2 | 119.5 | 110.4 | 74.9 |
| Sorbitol (nmol) | per U G6PDH | 23.1 ± 1.6 | 81.3 | 93.5 | 106.9 | 109.6 | 90.1 | 73.7 | 128.9* | 106.6 | 85.5 | 105.3 | 92.5 | 104.2 | 77.0 | 76.8 |
| Total (nmol) | per U G6PDH | 73.4 ± 4.6 | 101.1 | 120 | 96.2 | 94.1 | 44.8* | 45.2* | 96.0 | 84.6 | 86.0 | 107.6 | 72.0 | 82.0 | 78.1 | 69.2 |
| Protein (mg) | per U G6PDH | 0.066 ± 0.004 | 123 | 141 | 134 | 121 | 140 | 160 | 109 | 127 | 97 | 83 | 111 | 105 | 136 | 194 |
| Sperm motility | Total (%) | 72.2 ± 1.4 | 62.1* | 68.0 | 64.2* | 75.1* | 26.0* | 15.7* | 77.3* | 62.6* | 62.4* | 67.1* | 70.8* | 79.7* | 38.6* | 21.0* |
| Progressive (%) | 66.9 ± 2.4 | 31.5* | 29.7* | 29.8* | 34.3* | 53.2 | 11.8* | 33.0* | 36.8* | 41.2* | 48.4* | 54.5 | 55.8 | 22.9* | 50.0 | |
| Sperm migration | 1 cm (N) | 105.3 ± 16.1 | 36.5* | 31.0* | 32.7 | 28.9 | 15.2* | 14.1* | 55.1* | 25.8* | 63.8 | 54.8 | 49.9 | 42.6 | 18.9 | 13.1* |
| 3 cm (N) | 34.8 ± 6.4 | 23.4* | 16.9* | 14.5* | 4.0* | 1.3* | 8.0* | 32.5 | 10.0* | 40.7 | 29.1 | 9.6* | 9.5* | 16.3 | 3.4* | |
| CASA | VAP (μm s−1) | 78.6 ± 3.2 | 51.3 | 40.3* | 51.4 | 44.7* | 32.9* | 22.7* | 63.8 | 56.9* | 72.0 | 66.7* | 75.5 | 66.0* | 40.1* | 25.8* |
| VSL (μm s−1) | 68.0 ± 3.4 | 49.4 | 36.7* | 39.9 | 35.0* | 24.3* | 17.5* | 63.6* | 44.1* | 73.1 | 63.3* | 64.7 | 53.8* | 40.4 | 28.0* | |
| VCL (μm s−1) | 114.7 ± 4.2 | 45.8 | 42.7* | 44.8* | 62.7 | 54.5 | 32.3 | 79.3 | 83.1 | 68.3* | 68.4 | 64.9* | 66.8* | 48.6* | 25.9* | |
| LIN (%) | 59.3 ± 2.3 | 110.6 | 93.1 | 82.1 | 65.6 | 34.2* | 36.5* | 82.4 | 60.1 | 117.3 | 98.4 | 100.2 | 80.7* | 59.0* | 38.7* | |
| Sperm Tail | Straight (%) | 92.7 ± 1.9 | 50.3* | 51.9* | 63.3* | 54.0* | 74.8* | 89.3 | 39.1* | 52.9* | 68.0 | 44.2* | 60.4* | 62.4* | 79.6 | 78.9 |
| Sperm Head | Eosin-neg. (%) | 67.8 ± 3.4 | 77.2* | 88.7 | 69.1 | 70.3* | 31.1* | 16.2* | 83.3 | 73.4* | 58.1* | 62.5* | 63.6* | 71.1 | 44.6* | 28.9* |
| Mean absolute differences from internal controls (Protocol C: n = 5) | ||||||||||||||||
| Sperm tail | Coiled (%) | 4.9 ± 1.6 | 47.5* | 45.6* | 35.9* | 42.4* | 24.6 | 12.5 | 59.4* | 45.9* | 41.6 | 60.4* | 43.8* | 43.3* | 24.9* | 26.1* |
| Stump-tail (%) | 2.0 ± 0.8 | 5.3 | 5.2 | 3.7 | 3.6 | 3.4 | 3.1 | 4.2 | 4.4 | 1.40 | 1.90 | 1.30 | 3.2 | 5.30 | 5.1 | |
| Number of unchanged parameters | 4 | 3 | 5 | 3 | 3 | 2 | 6 | 2 | 7 | 4 | 5 | 3 | 4 | 2 | ||
A, multistep addition and removal of CPA; B single-step addition and removal of CPA; C, control: single-step addition and removal of basal medium alone; CASA, computer-aided sperm analysis; CPA, cryoprotective agent; DMSO, dimethyl sulphoxide; EG, ethylene glycol; THP, 1,1,1-Tris(hydroxymethyl)propane; G6PDH, glucose-6-phosphate dehydrogenase; GLY glycerol; LIN, linearity; PD2, propane-1, 2-diol; PD3, propane-1, 3-diol; THE, 1,1,1-Tris(hydroxymethyl)ethane; VAP, averaged path velocity; VCL, curvilinear velocity; VSL, straight line velocity;
*
significantly different from C. Treatments EG (A), GLY (A), PD2 (A, B), PD3 (A) are best for maintenance of functional parameters (fewest significant differences from control and better sperm quality).