Doug's+Notebook

4/19/13 ACTIVATION EXPERIMENT 2 (3 AGENTS)

Took the xy male that was diluted at 4/17 to test 3 different activating agents. NaCl, KCl and Urea find lowest activation osmolality and test the next three up, test 850 and test 1050 Percent Motility

VCL Linearity

4/17/13 XX MALE AND CRYO VIALS

Adam L. xx male was inspected today. It gave some milt after we probed it with some tubing. I collected as much as I could and checked the motility. It seemed to be less than 5% motile. Since it is valuable I tried to freeze it anyway. I did 10% DMSO and a 1:10 dilution of sperm since it had a low sperm count anyway. Feezing was 2.5-40c and -5 to -60 c but slower than 5 since it coulddnt freefall that fast at such low temps. Then punged into liquid nitrogen. and This was all in 2 ml vials. 5 of the xx and 5 of the sib xy fish.

4/15/13 ACTIVATION EXPERIMENTS 1 (TIME IN DILUENT)

Started day expecting to start activations experiments of nacl kcl and urea looking at differences in activation between the three and proving what I think is the mechanisms behind activation

Started with male 3D9.1BF0FF6797 Great runner tested low point of NaCl and KCl they were both low near 425/450 and I checked high activation for all three ad they were all good. I then ran out of vials and had to wait an hour and a half. I tested Urea low activation then and it turned out to be 550. I then checked the NaCl at 450 again to see if that worked and It didn't. Realized the low point of activation is moving up as time goes on in the diluent. made new diluted sperm and tested. Activation was low again. as time goes on it moved up. I tested the low point every 15 minutes for a few hours.


 * || 1115 || 1130 || 1145 || 1200 || 1215 || 1230 || 1245 || 100 || 115 || 130 || 145 || 200 || 230 || 300 ||
 * || 0 || 0.25 || 0.5 || 0.75 || 1 || 1.25 || 1.5 || 1.75 || 2 || 2.25 || 2.25 || 2.5 || 3 || 3.5 ||
 * 450 || 0.78 || 0 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * 475 ||  || 0.29 || 0.21 || 0.13 || 0.01 || 0 ||   ||   ||   ||   ||   ||   ||   ||   ||
 * 500 ||  ||   || 0.55 || 0.32 || 0.15 || 0.02 || 0.37 || 0.08 || 0.01 ||   ||   ||   ||   ||   ||
 * 525 ||  ||   ||   ||   ||   || 0.37 ||   ||   || 0.08 || 0.08 ||   ||   ||   ||   ||
 * 550 ||  ||   ||   ||   ||   ||   ||   ||   || 0.27 || 0.14 || 0.13 ||   ||   ||   ||
 * 575 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   || 0.25 || 0.1 || 0.09 || 0.02 ||
 * 600 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   || 0.11 ||

4/12/13 CRYO Results

Thawed one straw at a time by submerging in 38 Degree water for 7 seconds. Both ends clipped and sperm blown out of straw into vial.

Activated with 1025 NaCl and compared to o2 held sperm diluted right before activation test.

RESULTS One second analysis starting at 17 seconds (1649 Frames) CRYO % Motile VCL VAP VSL LIN WOB PROG BCF # Sperm 0.32222223 201.41206 145.68492 136.1147 0.93430877 0.72331774 4178.703 39.924072 90.0

0.2857143 193.04921 144.88832 136.8528 0.9445399 0.7505253 3109.4106 34.310303 112.0

0.29347825 190.58449 137.67798 130.08969 0.94488376 0.7223987 3573.4985 39.145588 92.0

0.2967033 195.36816 128.38692 117.48714 0.9151021 0.6571537 3857.47 44.18909 91.0

0.24719101 198.9595 144.48119 138.81833 0.9608055 0.7261839 4646.3364 45.419346 89.0

0.2173913 198.01698 144.51564 139.72893 0.96687746 0.7298144 4435.3433 45.72198 92.0

0.26041666 198.2731 142.71179 135.30034 0.948067 0.7197738 3938.5405 42.805584 96.0

0.24210526 202.1317 138.36954 133.19038 0.96257013 0.6845514 3853.9502 43.4659 95.0

0.2580645 193.97733 130.06035 124.4969 0.95722413 0.67049253 3402.8203 38.50422 93.0

0.24418604 186.4862 129.25243 125.1709 0.96842206 0.6930937 4191.272 44.785885 86.0

0.22727273 182.50075 123.35407 119.3162 0.96726596 0.67591 3894.0518 45.42995 88.0

0.21 169.09818 112.03896 107.4372 0.95892715 0.66256756 2374.915 37.884525 100.0

0.1724138 165.99487 109.09251 104.88536 0.9614351 0.6572041 3785.9634 42.6964 87.0

0.18181819 169.53815 111.3239 108.34566 0.973247 0.6566304 3405.6338 45.52725 88.0

FRESH % Motile VCL VAP VSL LIN WOB PROG BCF # Sperm 0.675 248.27249 176.32047 169.51651 0.96141136 0.71018934 5150.3223 46.272713 40.0

0.6666667 247.76485 179.76956 174.9844 0.9733817 0.72556525 5913.8247 48.432972 39.0

0.62222224 248.06783 180.17996 174.12808 0.966412 0.7263335 5301.003 46.566704 45.0

0.6363636 255.51927 184.14514 178.66812 0.970257 0.7206703 5648.428 49.52596 44.0

0.6363636 249.94519 184.24425 178.04875 0.9663735 0.73713857 5251.0767 46.43766 44.0

0.54285717 243.04266 177.41298 172.7597 0.9737715 0.7299664 5569.269 49.786663 35.0

0.63414633 231.3309 164.23952 156.38885 0.95219994 0.70997655 5086.8394 50.60281 41.0

0.6904762 230.26994 163.98431 156.26196 0.95290804 0.7121394 4234.4575 46.44054 42.0

0.6666667 242.2887 169.04257 162.91035 0.96372384 0.6976907 4585.852 46.71759 42.0

0.6511628 238.20525 171.9168 166.67258 0.96949565 0.72171706 4925.309 47.12716 43.0

0.61904764 235.33028 167.07532 162.79425 0.97437644 0.709961 5643.582 50.62719 42.0

0.60465115 230.96344 163.4367 159.14099 0.97371626 0.7076302 5447.8228 50.585625 43.0

0.627907 231.28789 163.92091 157.8805 0.9631504 0.7087311 5474.3438 52.895393 43.0

0.63829786 233.55646 163.38647 159.69337 0.97739655 0.6995588 5339.1797 51.682346 47.0

0.65217394 233.76428 159.65262 154.80782 0.9696542 0.6829641 3961.082 46.82538 46.0

VCL Comparison

% Motility Comparison



4/10/13 CRYO TRIAL TRIAL 1 Used a 1:100 Dilution of sperm from male fresh this morning 3D9.1BF102041D. Using the shaker at 40 rpm and 10 min I added 5 alequots of 495 ul cortlands to tray. added sperm 50 ul and shake till combined. Slowly added 10% DMSO Soln at 495 ul per 2 min for 10 min. Sucked with syringe into .25 ml straws and labeled sample 1,1,2,2 Label incorrect on 2,2 (correction not 1:10 but 1:100 Dilution) Sperm frozen at rate of 2.5 c/m from 4 to -40 degrees and 5 c/m to -50 then plunged into liquid nitrogen.

WE WILL SEE !!!

4-10-13 Cryoprotectant Prep Created 10% and 5% DMSO for use as cryoprotectant. Made new Cortlands soln at pH 7.8 Since DMSO must be added slowly to the diluted sperm solution I created 2x strength DMSO solutions 10% DMSO solution and 5% DMSO solution (v/v)
 * 50 ml solution ||  || 10% DMSO ||   ||   ||
 * || 45 ml || Cortlands || -25 || 20 ml cortlands solution ||
 * || 5 ml || DMSO 96% ||  ||   ||

For a 1:100 Solution For CASA 10 μl sperm and 495 μl pure corland's mixed together Slowly Drip 495 μl DMSO Solution into sperm dilution on shaker plate in cold room. Over 20 min For a 1:10 Solution For Fertilization 100μl sperm and 450 μl pure cotlands mixed together Slowly drip 450 μl DMSO soution into sperm dilution on shaker plate in cold room over 20 min
 * 50 ml solution ||  || 5% DMSO ||   ||   ||
 * || 47.5 ml || Cortlands || -25 || 22.5 ml cortlands solution ||
 * || 2.5 ml || DMSO 96% ||  ||   ||

4-5-13 Activation solution prep Created KCl NaCl and Urea solutions from stock 2 m soultions Using this workbook


 * || a || b ||  || c ||   || d || q || t || x || y || w || z ||
 * Label || Solution A || Solution B ||  || Volume of Soln.A ||   || Volume of Soln. B || Final Volume || Final Solution Osmolality || Extender || Desired mmol/kg || Volume of Cortlands || Volume of NaCl ||
 * DIUF || 0 || 1900 || KCl || 0.810 ||  || 0.188 || 0.997610551 || 358.0555556 || 277.5 || 350 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.797 ||  || 0.203 || 0.999654428 || 385.8333333 || 277.5 || 375 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.783 ||  || 0.218 || 1.001423774 || 413.6111111 || 277.5 || 400 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.767 ||  || 0.232 || 0.998665828 || 441.3888889 || 277.5 || 425 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.753 ||  || 0.247 || 1.000284192 || 469.1666667 || 277.5 || 450 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.740 ||  || 0.262 || 1.001721632 || 496.9444444 || 277.5 || 475 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.723 ||  || 0.276 || 0.999385918 || 524.7222222 || 277.5 || 500 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.710 ||  || 0.291 || 1.000723982 || 552.5 || 277.5 || 525 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.696 ||  || 0.306 || 1.00193394 || 580.2777778 || 277.5 || 550 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.680 ||  || 0.32 || 0.999908634 || 608.0555556 || 277.5 || 575 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.666 ||  || 0.335 || 1.001048493 || 635.8333333 || 277.5 || 600 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.650 ||  || 0.349 || 0.999229803 || 663.6111111 || 277.5 || 625 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.636 ||  || 0.364 || 1.000305344 || 691.3888889 || 277.5 || 650 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.622 ||  || 0.379 || 1.001297798 || 719.1666667 || 277.5 || 675 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.607 ||  || 0.393 || 0.999672741 || 746.9444444 || 277.5 || 700 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.593 ||  || 0.408 || 1.000616708 || 774.7222222 || 277.5 || 725 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.578 ||  || 0.423 || 1.001495327 || 802.5 || 277.5 || 750 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.563 ||  || 0.437 || 1.000026765 || 830.2777778 || 277.5 || 775 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.549 ||  || 0.452 || 1.000867595 || 858.0555556 || 277.5 || 800 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.535 ||  || 0.467 || 1.001655691 || 885.8333333 || 277.5 || 825 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.519 ||  || 0.481 || 1.000316206 || 913.6111111 || 277.5 || 850 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.505 ||  || 0.496 || 1.001074063 || 941.3888889 || 277.5 || 875 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.491 ||  || 0.511 || 1.001788478 || 969.1666667 || 277.5 || 900 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.476 ||  || 0.525 || 1.000557258 || 996.9444444 || 277.5 || 925 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.461 ||  || 0.54 || 1.00124695 || 1024.722222 || 277.5 || 950 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.447 ||  || 0.555 || 1.001900238 || 1052.5 || 277.5 || 975 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.432 ||  || 0.569 || 1.000761121 || 1080.277778 || 277.5 || 1000 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.417 ||  || 0.584 || 1.001393833 || 1108.055556 || 277.5 || 1025 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.403 ||  || 0.599 || 1.001995598 || 1135.833333 || 277.5 || 1050 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.388 ||  || 0.613 || 1.000935784 || 1163.611111 || 277.5 || 1075 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.374 ||  || 0.628 || 1.001520168 || 1191.388889 || 277.5 || 1100 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.359 ||  || 0.642 || 1.000519481 || 1219.166667 || 277.5 || 1125 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.344 ||  || 0.657 || 1.001087102 || 1246.944444 || 277.5 || 1150 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.330 ||  || 0.672 || 1.001629985 || 1274.722222 || 277.5 || 1175 || 0.1 || 0.9 ||
 * DIUF || 0 || 1900 || KCl || 0.315 ||  || 0.686 || 1.000690979 || 1302.5 || 277.5 || 1200 || 0.1 || 0.9 ||
 * ||  ||   ||   || (td-bd)/(a-t) ||   ||   || c+d || (yq-xw)/z ||   ||   ||   ||   ||


 * Desired mmol/kg || DIFU || NaCl ||
 * 350 || 41.861 || 8.050 ||
 * 375 || 41.358 || 8.700 ||
 * 400 || 40.616 || 9.300 ||
 * 425 || 40.094 || 9.950 ||
 * 450 || 39.370 || 10.550 ||
 * 475 || 38.834 || 11.200 ||
 * 500 || 38.124 || 11.800 ||
 * 525 || 37.575 || 12.450 ||
 * 550 || 36.876 || 13.050 ||
 * 575 || 36.318 || 13.700 ||
 * 600 || 35.628 || 14.300 ||
 * 625 || 35.063 || 14.950 ||
 * 650 || 34.380 || 15.550 ||
 * 675 || 33.808 || 16.200 ||
 * 700 || 33.131 || 16.800 ||
 * 725 || 32.554 || 17.450 ||
 * 750 || 31.883 || 18.050 ||
 * 775 || 31.300 || 18.700 ||
 * 800 || 30.634 || 19.300 ||
 * 825 || 30.047 || 19.950 ||
 * 850 || 29.385 || 20.550 ||
 * 875 || 28.794 || 21.200 ||
 * 900 || 28.136 || 21.800 ||
 * 925 || 27.542 || 22.450 ||
 * 950 || 26.945 || 23.100 ||
 * 975 || 26.290 || 23.700 ||
 * 1000 || 25.690 || 24.350 ||
 * 1025 || 25.038 || 24.950 ||
 * 1050 || 24.436 || 25.600 ||
 * 1075 || 23.786 || 26.200 ||
 * 1100 || 23.182 || 26.850 ||
 * 1125 || 22.534 || 27.450 ||
 * 1150 || 21.928 || 28.100 ||
 * 1175 || 21.283 || 28.700 ||


 * H20 || KCl || Desired mmol/kg ||
 * 41.56989915 || 8.4 || 350 ||
 * 40.91069114 || 9.05 || 375 ||
 * 40.25271995 || 9.7 || 400 ||
 * 39.59575205 || 10.35 || 425 ||
 * 38.93960924 || 11 || 450 ||
 * 38.28415316 || 11.65 || 475 ||
 * 37.62927475 || 12.3 || 500 ||
 * 36.97488688 || 12.95 || 525 ||
 * 36.3209191 || 13.6 || 550 ||
 * 35.79246231 || 14.3 || 575 ||
 * 35.13152031 || 14.95 || 600 ||
 * 34.47149435 || 15.6 || 625 ||
 * 33.81227401 || 16.25 || 650 ||
 * 33.15376593 || 16.9 || 675 ||
 * 32.49589067 || 17.55 || 700 ||
 * 31.83858014 || 18.2 || 725 ||
 * 31.1817757 || 18.85 || 750 ||
 * 30.52542656 || 19.5 || 775 ||
 * 29.86948851 || 20.15 || 800 ||
 * 29.21392286 || 20.8 || 825 ||
 * 28.55869565 || 21.45 || 850 ||
 * 27.90377693 || 22.1 || 875 ||
 * 27.24914015 || 22.75 || 900 ||
 * 26.59476177 || 23.4 || 925 ||
 * 25.99455137 || 24.1 || 950 ||
 * 25.33788599 || 24.75 || 975 ||
 * 24.68156338 || 25.4 || 1000 ||
 * 24.02555778 || 26.05 || 1025 ||
 * 23.36984593 || 26.7 || 1050 ||
 * 22.71440678 || 27.35 || 1075 ||
 * 22.05922126 || 28 || 1100 ||
 * 21.40427204 || 28.65 || 1125 ||
 * 20.74954333 || 29.3 || 1150 ||
 * 20.0950207 || 29.95 || 1175 ||
 * 19.44069098 || 30.6 || 1200 ||


 * DIFU || UREA || Desired mmol/kg ||
 * 40.48052754 || 9.4 || 350 ||
 * 39.83272138 || 10.15 || 375 ||
 * 39.17118872 || 10.9 || 400 ||
 * 38.33329138 || 11.6 || 425 ||
 * 37.66420959 || 12.35 || 450 ||
 * 36.98608161 || 13.1 || 475 ||
 * 36.16929592 || 13.8 || 500 ||
 * 35.4861991 || 14.55 || 525 ||
 * 34.79669698 || 15.3 || 550 ||
 * 33.9954317 || 16 || 575 ||
 * 33.30242464 || 16.75 || 600 ||
 * 32.51149016 || 17.45 || 625 ||
 * 31.81526718 || 18.2 || 650 ||
 * 31.11488992 || 18.95 || 675 ||
 * 30.33363704 || 19.65 || 700 ||
 * 29.63083542 || 20.4 || 725 ||
 * 28.92476636 || 21.15 || 750 ||
 * 28.15133824 || 21.85 || 775 ||
 * 27.44337973 || 22.6 || 800 ||
 * 26.73278457 || 23.35 || 825 ||
 * 25.96581028 || 24.05 || 850 ||
 * 25.25370316 || 24.8 || 875 ||
 * 24.5394239 || 25.55 || 900 ||
 * 23.77786291 || 26.25 || 925 ||
 * 23.06234752 || 27 || 950 ||
 * 22.34501188 || 27.75 || 975 ||
 * 21.58805606 || 28.45 || 1000 ||
 * 20.86969165 || 29.2 || 1025 ||
 * 20.1497799 || 29.95 || 1050 ||
 * 19.39678921 || 30.65 || 1075 ||
 * 18.67600839 || 31.4 || 1100 ||
 * 17.92597403 || 32.1 || 1125 ||
 * 17.20435509 || 32.85 || 1150 ||
 * 16.48149924 || 33.6 || 1175 ||
 * 15.73454894 || 34.3 || 1200 ||

__ CASA Setup __

Issues with video where sperm have too much background noise when trying to process. Removed all filters and added thick dark paper to creat a faux darkfield effect to make sperm appear white on black background. 10x objective with 97 fps video camera attached via ethernet to toughbook with labview view capture software.

__Sperm count and pH of seminal fluid__ Aug 27 3D9.1BF1312CB5 7.8 Aug 28 3C8.000041B2E3 8.07 Aug 28 3D9.1BF1312B2D 7.8 Sept 6 3C8.000041B2E3 7.75 24.8 B 26.5 B Sept 6 3C8.000041AC84 7.52 24.6 B 23.8 B Sperm count is taken by hemocytometer reading of 1:1000 diluted sperm under 10x objective with phase contrast axioscope
 * Average || 24.06 || Billion/mL ||

Osmolality Data of Ovarian fluid and Seminal fluid
 * Ovarian Ph Average ||  ||   ||   ||   || 8.07 || pH ||
 * Ovarian fluid Osmolality || Sample a || Sample b || Average ||  ||   ||   ||
 * || 346 || 349 || 347.5 ||  ||   ||   ||
 * || 363 || 359 || 361 ||  ||   ||   ||
 * || 351 || 352 || 351.5 ||  ||   ||   ||
 * || 367 || 364 || 365.5 ||  ||   ||   ||
 * || 355 || 351 || 353 ||  ||   ||   ||
 * || 334 || 330 || 332 ||  ||   ||   ||
 * || 344 || 349 || 346.5 ||  ||   ||   ||
 * || 334 || 330 || 332 ||  ||   ||   ||
 * || 336 || 336 || 336 ||  ||   ||   ||
 * || 336 || 334 || 335 ||  ||   ||   ||
 * || 328 || 330 || 329 ||  ||   ||   ||
 * || 369 || 371 || 370 ||  ||   ||   ||
 * || 338 || 337 || 337.5 ||  ||   ||   ||
 * || 339 || 341 || 340 ||  ||   ||   ||
 * || 338 || 334 || 336 ||  ||   ||   ||
 * || 291 || 396 || 343.5 ||  ||   ||   ||
 * || 323 || 330 || 326.5 ||  ||   ||   ||
 * || 333 || 332 || 332.5 ||  ||   ||   ||
 * || 336 || 335 || 335.5 ||  ||   ||   ||
 * || 341 || 337 || 339 ||  ||   ||   ||
 * || 335 || 333 || 334 ||  ||   ||   ||
 * || 319 || 319 || 319 ||  ||   ||   ||
 * || 330 || 330 || 330 ||  ||   ||   ||
 * || 337 || 339 || 338 ||  ||   ||   ||
 * || 344 || 335 || 339.5 ||  ||   ||   ||
 * || 328 || 330 || 329 ||  ||   ||   ||
 * || 305 || 302 || 303.5 ||  ||   ||   ||
 * || 354 || 338 || 346 ||  ||   ||   ||
 * || 334 || 332 || 333 ||  ||   ||   ||
 * || 340 || 331 || 335.5 ||  ||   ||   ||
 * || 302 || 304 || 303 ||  ||   ||   ||
 * || 328 || 333 || 330.5 ||  ||   ||   ||
 * || 337 || 338 || 337.5 ||  ||   ||   ||
 * || 343 || 342 || 342.5 ||  ||   ||   ||
 * || 334 || 324 || 329 ||  ||   ||   ||
 * || 332 || 336 || 334 ||  ||   ||   ||
 * || 325 || 326 || 325.5 ||  ||   ||   ||
 * || 333 || 337 || 335 ||  ||   ||   ||
 * || 357 || 356 || 356.5 ||  ||   ||   ||
 * || 326 || 327 || 326.5 ||  ||   ||   ||
 * || 326 || 325 || 325.5 ||  ||   ||   ||
 * || 321 || 322 || 321.5 ||  ||   ||   ||
 * || 326 || 325 || 325.5 ||  ||   ||   ||
 * || 336 || 333 || 334.5 ||  ||   ||   ||
 * || 323 || 325 || 324 || Average || 335.7333333 || mOsm/L ||
 * Seminal fluid Ph || 7.8 ||  ||   ||   ||   ||   ||
 * || 8.07 ||  ||   ||   ||   ||   ||
 * || 7.8 ||  ||   ||   ||   ||   ||
 * || 7.75 ||  ||   ||   ||   ||   ||
 * || 7.52 ||  ||   ||   ||   ||   ||
 * || 7.62 ||  ||   || Average || 7.76 || pH ||
 * Seminal fluid osmolality || 336 || 334 || 335 ||  ||   ||   ||
 * || 324 || 327 || 325.5 ||  ||   ||   ||
 * || 331 || 334 || 332.5 || Average || 331 || mOsm/L ||

__4-21-12__

Fertilized samples of eggs kept overnight in 6 well trays in 6 °C Sample A washed in cortlands and held overnight in it Sample B Kept in normal ovarian Fluid Held overnight and fertilized with 250 μl 1:10 sperm from day previous activated with 50 ml seawater 9:30 pm Fert check Kept in wash Fertilized Not fertilzied A 41 69 B 27 66 C 68 85 No Wash A NA B 58 79 C 67 83 Results: Egg was has not significant impact on fert rates or symetry

__4-20-12 Diluent dH2O and seawater__ 200 ml dH2O + 100 ml Seawater from local head tank fitered with vacumn container put in freezer to chill quickly

.5 ml from both sperm samples taken 4-18-12 diluted with mixture made above 9 ml

1:10 dilution Ovarian fluid pH average 8.07 Recorded from 81 female samples from spawning pulse 1-2 Egg fert and wash test Egg symetry and wash both poor
 * Modified Corlands Sperm Extender Solution Formula ||  ||
 * Desired Volume in L ||  ||   ||   ||   ||
 * 0.5 || L ||  ||   ||   ||
 * ||  || g/L || 0.5L ||   ||
 * NaCl || 124.1 mM || 7.25 || 3.625 || g ||
 * KCl || 5.1 mM || 0.38 || 0.19 || g ||
 * CaCl2 2H2O || 1.6 mM || 0.235 || 0.1175 || g ||
 * MgSO4 7H2O || 1 mM || 0.246 || 0.123 || g ||
 * Glucose || 5.6 mM || 1 || 0.5 || g ||
 * HEPES || 20 mM || 4.77 || 2.385 || g ||
 * NaOH || 1 M |||| Add to desired pH ||  ||
 * NaOH || 1 M |||| Add to desired pH ||  ||

__4-18-12 PVA Solution__ 1g PVA 99-100% Crystalline Power 85 ml dH2O Let cool add DH2O to 100 ml Hold at 4 Degrees

Coat ibidi 6 well slides with pva pipet 1% PVA soln into wells on one side of slide Tap slide while angling slide so soln fills channel pipet 1 ml dH2O through slide to rinse blow out with air let dry

ibidi 80601 microwell slide VI 6 channel uncoated

Collected sperm from two males and kept in 4degree with oxygen in whirlpak bag