Use and accuracy of instruments to estimate sperm concentration: Pros, Cons & Economics

Use and accuracy of instruments to estimate sperm concentration: Pros, Cons & Economics R. V. Knox, S. L. Rodriguez-Zas, S. Roth, and Kelly Ruggiero Department of Animal Sciences, University of Illinois, Urbana Importance of Sperm Concentration for AI AI success can depend upon sperm quality at collection, sperm age when inseminated, insemination volume, and AI times relative to estrus. However, in addition to these factors, another essential component of AI effectiveness is the number of sperm inseminated. In fact, concentration of sperm inseminated, may actually compensate for many deficiencies involving the technology when using AI, when conditions are less than optimal. Therefore, properly estimating the concentration of the boar ejaculate is critical for efficient boar use and for influencing the effectiveness of the AI technology to optimize farrowing rate (FR) and litter size (LS). Two billion sperm appears to optimize conventional AI for LS and FR when semen that is less than 36 h old, is administered within 28 h prior to ovulation 1. To further illustrate this, Watson and Behan 2 reported that when using conventional AI, 1 billion sperm resulted in lower farrowing rates and smaller litter sizes when compared to 2 or 3 billion sperm, (AI at 0 and 24 h, and semen used within 48 h of collection). However, when bypassing the cervix and much of the uterus with surgical insemination, 1 versus 3 billion had no effect when insemination was performed at < 10 h before ovulation 3. Apparently, the importance of sperm concentration used in an AI dose becomes clear when it was reported that the presence and numbers of sperm found in the oviduct following AI was only observed when >50-500 million sperm /cc (1-10 billion/dose) were used 4. However, Steverink et al. 5 indicated that when semen doses contained 1-6 x 10 9 sperm/dose, and were used within 36 h, the effect on fertility and accessory sperm counts were not apparent. Yet, as dose of sperm increased, and as the insemination to ovulation interval decreased, more accessory sperm were found bound to the eggs. This, and other data, suggests that in conditions of limited fertility, reproductive efficiency may depend on sperm concentration. From these studies it is clear that the sperm used in conventional AI must supply enough sperm to allow establishment of a viable sperm reservoir. The high numbers of sperm in a dose may help to keep a reservoir functional and compensate for increased intervals from insemination to ovulation. In a critical test of the effect of low sperm numbers (or viable sperm), the low dose technique using surgical insemination into the tip of the uterine horn has shown that 1 million to 2 billion sperm inseminated in 0.5 ml 3 results in no differences in FR or LS were observed when 10 million or more sperm were used for FR and LS. However, a 50% reduction was observed when 2 million or less was used. Another test of the effects of limited viable sperm occurs following freezing and thawing. In fact, more sperm must be used (5 billion) to achieve the same reproductive rates when compared to non-frozen-thawed sperm. Additionally, insemination must occur even closer to time of ovulation (within 4 hours of ovulation 6 ). In most cases, when using frozen thawed sperm, the 5 billion sperm used for AI, are intended to compensate for loss of fertile sperm, and provides ~1.5 billion motile sperm 7. Collectively, these studies indicate that when using conventional AI, 2 billion fertile sperm 20

can reliably result in good fertility. However, when using compromised sperm numbers, fertility is at risk. It is clear that a quick and reliable method to evaluate sperm concentration in an ejaculate for use in AI is required. The two methods that are commonly used for evaluation of sperm concentration include cell-counting chambers used with a light microscope, and analysis by photometry. Gross Evaluation of Sperm Concentration Sperm concentration greater than 200 million cells per/ml is the minimal concentration recommended for extension. Ejaculates should initially be evaluated based on weight (volume), opacity (clarity), color, odor, and turbidity (swirling) before further processing. A visual assessment the opacity or the amount of light that passes through an ejaculate is an indication of the number of sperm cells. Less transparent samples therefore contain more sperm cells. The sperm rich fraction is creamy in appearance and allows little light pass though. The sperm poor fractions are more watery in appearance, are more transparent, and have fewer cells. Ejaculate turbidity is another indicator for sperm cell numbers. It appears when the sample is gently rotated and a swirling motion can be seen in the liquid. For an example of this, let the sample set long enough for sperm cells to settle to the bottom of the container and then gently rotate the sample. Semen Sub-Sampling for Evaluation Before any further technical evaluation is performed, sub-sampling of the ejaculate should be made with a well-mixed sample. Gentle swirling is required since sperm cells settle to the bottom with time. It should be mentioned that shaking or swirling too vigorously could cause damage to the sensitive attachment region for the sperm head and tail. Estimating Sperm Concentration Using the Hemacytometer Evaluation of a semen sample with the hemacytometer (blood cell counting chamber) provides a method for accurate determination of sperm cell concentration. An accurate estimation of concentration will allow extension rates that optimize boar utilization and fertility of extended semen. The materials needed for this routine procedure should include a good quality microscope (~$1500), and a hemacytometer (~$100/ea). The sample dilution system can include a red or white blood cell dilution system (Unopette, ~1.00/ea) or a handpipette system. The Unopette white blood cell dilution system is desirable since no additional equipment is needed, sperm cells are killed, and pipetting and dilution errors can be minimized. In this system, a sub-sample from the ejaculate is taken by mixing the ejaculate well and placing 2-3 drops on a microscope slide. The Unopette system allows a 10-20 µl sample to be obtained with a Unopette capillary pipette. The semen sample in the capillary tube is then added to the Unopette dilution chamber and mixed. The diluted semen sample is then applied to both chambers of hemacytometer. After ~3-5 minutes, the sperm cells settle onto the glass and counting of sperm at 200-400 magnification can begin. Since sperm are almost transparent under light microscopy, a phase-contrast scope can facilitate sperm visualization. When using a standard light microscope, the iris diaphragm should be closed to improve sperm visualization. Sperm are counted in 5 diagonal squares. Sperm heads are not 21

counted if they touch the right or bottom triple lines of the squares (Figure 1). These sperm are not included since they contribute to over-estimation of the concentration when multiplication is performed. Both chambers of the hemacytometer are counted. Sperm numbers from each side should be within 10% of each other. If not, clean the hemacytometer and repeat sample addition (it is not necessary to repeat the dilution step). When sperm counts are within 10%, average the two numbers. If a 1:100, or a 1:200 Unopette dilution was used, simply add seven zeros to get sperm concentration/ml. This is a simplified way to determine concentration (for calculation example, see Appendix). Accuracy of the Hemacytometer The accuracy of the hemacytometer has a built in measurement system, with dual counting chambers. The objective is to arrive at count that is within 10% from one side to the other. However, this only estimates the intra-sample error from counting. To evaluate error from pipetting and counting, evaluate the count from two independent (inter-assay) counting events of the same sample. An evaluation of a raw semen sample diluted 1:100 with standard procedures compared to a count of semen from the spectrophotometer dilution range (1/5 to 1/50) can be used for providing counts in the optimal ranges. In our evaluation of the hemacytometer system (n= 60), intra-assay errors were less than 10% and inter-assay error estimation of sperm concentrations indicated that values were not statistically different and were within 5% of each other (188 versus 196 ± 12 million sperm/ml). Estimating Sperm Concentration Using the Photometer or Spectrophotometer Spectrophotometry is a measure of transmitted light as a function of wavelength through a reference liquid or blank. When a beam of light (wavelength), is focused on a semen sample, light is absorbed, scattered, or transmitted, depending upon the number, size, shape and opacity of the particles. The wavelength chosen is not as critical with white suspensions, but wavelengths for sperm appear most sensitive in the range of 550 to 576 nm (Foote). The equipment measures the relative amount of light transmitted, which can be displayed (% Transmittance, T) or converted into absorbance (-log T, A), which is directly proportional to concentration (Absorbance = absorptivity of sample x wavelength x concentration) 8. For semen evaluation, spectrophotometers (variable wavelengths) or photometers (single wavelengths) can be used. These single beam light sources can read in transmittance (0-125%), absorbance (-0.1-2.0), or concentration (0 999) units. The equipment is designed to minimize stray light and maintain narrow bandwidths. A sample of diluted semen (range: 1:2 to 1:200) is placed in a sample container inside the machine. A tungsten lamp (which has the features of high energy and longevity) generates light in the visible light range (360-700 nm). The radiant energy of the lamp passes through light filters and through a lens, which concentrates the light passing through the sample cuvette or tube (a high quality plastic or glass sample holder, which typically transmits >75% of the light through a blank sample). On the opposite side of the sample, a detector (photodiode) produces an electrical current proportional to the amount of light transmitted. This signal is then amplified and converted for display. 22

Photometers and spectrophotometers are priced between $1,500 to $6,000. The equipment may read in light absorbance units or % light transmittance. Many of the commercially available photometers have predetermined curves that calculate the concentration of sperm/cc while others provide a reading that is used to convert the reading to sperm/cc. The accuracy for each machine is based on a standard curve. Taking multiple semen samples and diluting them (1:10, 1:25, 1:50, 1:100, and 1:200) generates the standard curve. The transmittance or absorbance is then measured for each sample. A sperm cell count can then be performed on the original semen sample to determine the actual number of sperm cells/cc. A linear curve can be generated by dividing the actual known cell concentration by each of the dilutions (i.e. 5.0 x 10 8 /cc divided by 200 (e.g. 1:200 dilution) = 2.5 x 10 6 /cc. This is repeated for each dilution of each ejaculate sample. Each sample dilution is associated with an absorbance reading and establishes a relationship with sperm cell numbers (linear regression). A regression equation is then used to predict sperm cell numbers based on the absorbance or transmittance reading. There is inherent variation around any reading and only repeated sampling of different ejaculates around a photometer reading will improve confidence in the estimate of the sperm cell numbers. Unfortunately, light scattering is affected by seminal plasma within and between boar ejaculates, which may account for up to -19% to +30% errors in photometer estimations of actual sperm concentrations. Some units require dilution prior to reading while others do not. Readings near the upper and lower limits of detection (<10% and >90% for transmittance, or <0.2 and >1.8 absorbance) have high degrees of reading variation (low accuracy). This is because of the combination of too few or too many cells, and the detection of other factors that can alter the passage of light through a semen sample. This may actually become exaggerated at high semen dilution rates. Manufacturer instructions should be followed and calibration and accuracy of the equipment should be performed as recommended. Accuracy in Estimating Sperm Concentration Most of the equipment can resolve to.001 absorbance units, and have a precision of.005 absorbance units. The accuracy for spectrophotometers is reported to approximately ±.003 absorbance units and typically for photometers to.01 absorbance units (± 2% of the true value). Early research evaluating boar semen at concentrations of 54 to 287 x 10 6 sperm/ml using a photometer, indicated that the average error was ± 16.2%. Comparison of the accuracy of the spectrophotometer to the hemacytometer has been performed in many species including honeybees, buffalo, fish, turkeys, cattle, horses, sheep and swine. Generally measurements have all been performed in the 500-600 nm wavelength range and units measured were transmittance or absorbance. Paulenz et al. 9 compared the spectrophotometer (1:2 dilution; SpermaCue, HemoCue AB, Angelholm, Sweden), hemacytometer (1:200 diltuion), and Coulter counter (Coulter Electronics, Luton, England). There findings indicated that the hemacytometer was the most variable (12% CV, compared to the spectrophotometer (2.9%), and the Coulter counter (2.3%). Yet all three appeared to be highly related, and in an evaluation of the accuracy of the three methods, it was clear that both the spectrophotometer and the Coulter counter over- and under estimated the values at high and low concentrations. Correlation between concentrations and transmittance in almost all cases was high and greater than -0.96. In our study involving 29 boar ejaculates, we also observed a high correlation of concentration with transmittance (-0.98). However, the 23

primary factors limiting this relationship are the precision of pipetting, the reliability of the reagents, and the optimal transmittance/absorbance ranges for the equipment. The Twyman- Lothian curve (Spectronic 401) indicates the percent error in concentration as a function in percent transmittance (Figure 2). The curve indicates that when reading outside of the optimal rage (20 to 50 % T or 0.3-0.7 A, the estimates for concentration can be less precise 10. Other sources for variation can include particulate matter in the suspension, scratched containers, precipitation of sperm cells or other dissolved substances. Generating a Standard Curve. A useful way for quickly predicting the sperm concentration based on transmittance or absorbance is through construction of a standard curve, which plots the absorbance (or transmittance) of samples on the y-axis with the known concentration on the x-axis, and determining the useful portion with the best fit. Validation for the useful range for the spectrophotometer can be determined by taking a sample of known concentration and diluting it from 1:25 to 1:250. The results of the known concentration divided by the dilution rate can be plotted against the transmittance. This figure can be used to evaluate the predictive range for the machine (Figure 3). As can be seen, the data is similar to that for the Twyman-Lothian curve and arrows indicate the optimal accuracy ranges based on the previous reports. However, if plotting the straight line regression, it appears that the best fit for the dat actually lies between 35 to 80% transmittance, suggesting that the Twyman Lothian findings for some substances, may not apply to semen. Comparison of Equipment Evaluation of different photometer equipment has been performed and for two instruments using the same bandwidth, the calibration curves were not different. In other studies, triplicate assessment of sample counting errors was estimated at 12.3% (CV) for the hemacytometer and 2.9% for the spectrophotometer. In our lab, we evaluated a spectrophotometer (Spectronic 401, Spectronic Instruments, Inc. Rochester, NY) versus a photometer (Hyperion Micro-Reader I, Hyperion, Inc., Miami, FL) by dilution levels from 1:5 to 1:500 using 29 boar ejaculates. The data suggested that for each machine, the dilution range could influence the optimal reading range and for the Micro-reader I, the optimal range based on previously published reports, was 1:20 (0.3 to 0.7 A0), and for the Spectronic 401 was 1:15. At the same wavelength of 576 nm, the patterns were very similar and gave similar predictive values (Figure 4). The absorbance values resulting from the actual hemacytometer counts of the semen samples indicated that the values were influenced by boar, but not by equipment. For all samples, the readings, although nearly identical in pattern, produced different absolute levels. In fact the Spectronic 401 always produced higher transmittance readings. In our study, the average concentration evaluated was 179.6 x 10 6 sperm/ml (± 83.0, SD), and all samples ranged from 47.5 to 375 6 x 10 6 sperm/ml In this study, the correlation between the transmittance and absorbance was 0.98 and transmittance was defined as = e (-abs). It is clear then, that regardless of the equipment used, the same degree of reliability could be expected between machines. However, absolute values are expected to be different. 24

Optimal Dilution Range The ability to perform a single dilution that would provide optimal readings for any given ejaculate within the limits of the spectrophotometer would be ideal. The dilution range from sperm concentrations that produced the steepest slope, with the least variation, and accounts for most of the variation in absorbance (or transmittance) would be the best for predicting the concentration from the absorbance reading. For evaluation of the repeatability of certain dilution ranges, an evaluation of 8 separate boar ejaculates at dilutions of 1/50, 1/25 and 1/10 was performed in triplicate. The average readings for the Micro-reader was 0.17, 0.34, and 0.76 O.D. units, respectively. The standard errors as a percentage of the mean for each dilution were similar, and for the 1:10 was 5.1%, for the 1/25 was 6.6%, and for the 1/50 was 6.8%. This indicates, that the lower dilution rates may be expected to be less variable. Our evaluation of different dilution ranges using a spectrophotometer (Figure 5) and a photometer (Figure 6) and how they relate to % transmittance is shown. Both pieces of equipment produced nearly identical results, and the dilution ranges at 1:50 or greater (150 to 1:500) produced slopes that did not significantly differ from 0 (P >.05), and therefore would make poor estimates for concentration. The slopes were consistently different from 0 when semen was diluted less than 1:50 (P<.05). The dilution range that explained the greatest amount of variation in transmittance by concentration was the 1:5 dilution for both the Micro-reader I and the Spectronic 401 (P<.0001) compared to all other dilutions. This dilution had the steepest slope, the smallest standard error of the slope and was most significant. In fact a, 1 million change in sperm cell concentration would alter the transmittance.0006%. In the range of concentrations tested, 50 to 360 million sperm/cc, the transmittance (45% to 20%) or absorbance, was a good predictor for sperm concentration. In an assessment of the accuracy of certain dilutions over the others, at each dilution we compared the proportion of the rankings that were similar between the photometer and the spectrophotometer (samples arbitrarily ranked high, medium, or low based on transmittance) for all boars. The percent correct rankings were 1:5, 86%, 1:10 (80%), 1:15 (80%), 1/20 (90%), and 1/25 (66%). Although not statistically tested, this does indicate high repeatability and confidence at the lower dilution rates, and matches the high predicting equation accuracy at the 1:5 dilution. It should again be mentioned, that Foote 8 and the Twyman-Lothian curve (Spectronic 401) 10 have indicated that the optimal range for transmittance and absorbance are narrow and accurate only in certain ranges (30-50% T, and 0.3 to 0.7 A). Since from Figure 5 and 6, values out of the optimal range provide the best estimators for concentration, and values lying within the suggested optimal range are poor estimators, an overall conclusion that should be drawn is that each operation, machine, and dilution series should be evaluated by a standard curve which was generated within lab, since not all conditions can be expected to be optimal from one lab to the next. Support for this comes from the fact that the Spectronic 401 consistently reads at a lower absorbance than the Micro-reader. Perhaps this is not surprising, since the Spectronic would be expected to reduce the level of outside light interference. However, perhaps another concern should address the expected or actual levels of boar sperm concentration observed in the filed today compared to past decades. It is well noted that semen samples evaluated over the last four decades have ejaculates in the range of 40-60 25

billion/ejaculate and a concentration of 150-350 million/ml. However, newer data 11 indicates an average ejaculate output of 82 billion when boars are collected about once per week. Based on estimates then, the expected volume might be ~220 ml and average sperm concentration of ~370 million sperm/cc. It is possible, that lower collection frequencies, selection for increased testes size, greater maturity, better health and nutrition, have all lead to a dramatic increase in average sperm output from boars in stud. If this is the case, perhaps new standard curves (and higher dilution rates) will be needed to evaluate the increased sperm concentration. One example of this comes from Paulenz et al. 9 who observed that when using the SpermaCue at 1:2, the optimal reading range was 60-220 x 10 6 sperm/ml However, in their study, more than 83% of all boar ejaculate samples were of greater concentration than the maximum recommended reading value of 259 x 10 6 sperm/ml In fact, the average concentration for the 1-3 year old boars was ~ 410 x 10 6 sperm/ml This lends further support to the notion that conditions for sperm production have been greatly improved over the last decades. Semen Extension In gross semen extension, criteria such as volume, opacity, turbidity, and color are used to evaluate whether to extend the sample. In this system, no microscope or photometer is used to determine sperm concentration. Extension rates instead, are based on previous knowledge of the normal ranges for total sperm output in an ejaculate. Since most boars that are healthy and have not been overused, ejaculate in the range of 20-60 billion sperm, dilution rates between 1:4 and 1:10 can be used. For example, a 1:4 dilution rate, would in theory, provide semen doses with 5-15 billion cells per dose, while a 1:10 dilution would produce doses containing 2-6 billion sperm. Obviously, there are inherent flaws in this system, but "blind" dilutions within this range can generally produce acceptable fertility results. However Rozeboom et al. 12 reported that seminal plasma should be at least 10% of the extended semen to take advantage of its multiple effects. This typically limits the extension range based on the original collection volume (100-550 ml) and the total motile sperm in the ejaculate (40-120 x 10 9 sperm) and the need to keep seminal plasma between 10-20%. REFERENCES Nissen, A. K., N. M. Soede, P. Hyttel, M. Schmidt, and L. D Hoore. 1997. The influence of time of insemination relative to time of ovulation on farrowing frequency and litter size in sows, as investigated by ultrasonography. Theriogenology 47:1571-1582. Watson, P., and J. Behan. 2001. Deep insemination of sows with reduced sperm numbers does not compromise fertility: A commercially based field trial. IMV International Swine Reproduction Seminar, Minneapolis, MN. Rath, D., C. Krueger, and L.A. Johnson. 2000. Low dose insemination in the pig.. In: Boar Semen Preservation IV. L.A. Johnson and H.D. Guthrie (eds.). Allen Press, Inc., Lawrence, KS. 26

Baker, R.D., P.J. Dziuk, and H. W. Norton. 1968. Effect of volume of semen, number of sperm and drugs on transport of sperm in artificially inseminated gilts. J. Anim. Sci. 27: 88-93. Steverink, D. W., N. M. Soede, E. G. Bouwman, and B. Kemp. 1997. Influence of insemination-ovulation interval and sperm cell dose on fertilization in sows. J. Reprod. Fertil. 111:165-171. Waberski D., K. F. Weitze, T. Gleumes, M. Schwartz, T. Willmen and R. Petzoldt. 1994. Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen. Theriogenology 42:831-840. Hofmo, P.O., and I. S. Grevle. 2000. Development and commercial use of frozen boar semen in Norway. In: Boar Semen Preservation IV. L.A. Johnson and H.D. Guthrie (eds.). Allen Press, Inc., Lawrence, KS. Foote, R.H. 1972. How to measure sperm cell concentration by turbidity (optical density). Proceedings of the Fourth Technical Conference on Artificial Insemination and Reproduction. N.A.A.B. Paulenz, H., I. S. Grevle, A. Tverdal, P. O. Hofmo, and K. Andersen Berg. 1995. Precision of the Coulter counter for routine assessment of boar-sperm concentration in comparison with the haemocytometer and spectrophotometer. Reprod. Dom. Anim. 30:107-111. Spectronic 401, Spectronic Instruments, Inc. Rochester, NY. Rutten, S. C., R. B. Morrison, and D. Reicks. 2000. Boar stud production analysis. Swine Health and Prod. 8:11-14. Rozeboom K. F., M. H. T. Troedsson, G. C. Shurson, J. D. Hawton, and B. G. Crabo. 1997. Late estrus or metestrus insemination after estrual inseminations decreases farrowing rate and litter size in swine. J. Anim. Sci. 75:2323-2327. 27

APPENDIX Quality Control Procedures for Evaluation of Semen Concentration Exercise 1 Determine sperm concentration of sample A. The actual formula is ((sperm in 5 squares x 5; (or total sperm in 25 squares)) x dilution rate (100 or 200) x hemacytometer chamber volume (10,000 or 10 4 ) = sperm/cc) ). Example: (25 sperm average (in 5 squares) x 5) x 200 (dilution factor) x 10 4 = 250,000,000 sperm/cc. The total time required for this procedure is about 10 minutes per sample. Errors typically occur with this procedure due to pipetting error and improper chamber filling (under or over-filling). Exercise 2 Calculate repeatability of hemacytometer reading. Intra-dilution (both sides of a single chamber should be within 10%). Inter-dilution error between samples, of sample C at 1:100 (Unopette) versus 1:20 of the diluted sample (typical of a spectrophotometer sample). Exercise 3 Determine spec repeatability (boar A and B) at a single dilution in triplicate Boar A 1 2 3 Boar B 1 2 3 The intra-assay can be determined. The CV is a measure of variation of the triplicates and should be less than 20%. The CV is determined by dividing the SD by the mean. Exercise 4 A standard curve can be generated within a single day or over a period of time. What is needed is for a variety of samples that differ in concentration, the range covers the typical or expected range for samples that the lab should expect to evaluate over time. The blank should be 3% sodium citrate, but 0.9% sodium chloride can be used. It is possible to find the optimal value for the equipment, boar ejaculate concentration, that different ranges (two) dilution levels may need to be evaluated (for example 1:5 compared to 1:20). Test for dilution range (1:5 and 1:20) Boars A-E A regression line can be generated to predict the concentration based on the values of transmittance or absorbance (or optical density). In an EXCEL spreadsheet, create two columns, the 1 st containing the concentration and the second containing the spectrophotometer values (in this case, transmittance). Select the function f x icon to select the regression function to determine the slope and intercept and use the x and y coordinates to create the estimates. The regression equation can be generated by the formula y = b 1 x + b 0 where b 1 is the slope, b 0 the intercept, and x the spectrophotometer value. One equation is generated for 1:5 (boars A-E) and another at 1:20 (Boars A-E). A correlation coefficient can be generated to determine which regression equation accounts for the most variation ( r = 0.0-1.0) in absorbance (or transmittance) using the sperm concentration. 28

Column 1 Column 2 b 1 b 0 Y % Transmittance Conc. (x10 6 ) slope Intercept Predicted Value 29

Exercise 5 Determine Extension Rate 1. Sperm cells/cc x % motile x % normal x ejaculate volume = total sperm. 2. e.g. 60 billion sperm x 90% motile x 95% normal x 150 cc = 51 billion sperm. 3. To get a desired 3 billion sperm/80cc insemination dose: 340 x 10 6 sperm/cc (determined sperm concentration) x 150 cc (ejaculate volume) = 51 x 10 9 total sperm. 4. 51 x 10 9 sperm 3.0 x 10 9 sperm/dose = 17 doses 5. Multiply doses (17) x volume of semen dose (80 cc) = 1360 cc final volume. 6. 1360 cc - 150 cc = 1210 cc 7. Add 1210 cc of extender to 150 cc semen sample. 8. Gently aliquot 80 cc of extended semen into 17 bottles, tubes, or bags for 3.0 x 10 9 sperm/dose. 9. Test with counting under microscope (usually dilution needed at 1:5-1:50) 30

Economics of concentration errors In this scenario, a boar is collected and produces 200 ml of semen. The actual sperm concentration is 250 x 10 6 sperm/ml. However, in five different scenarios, errors ± 25-50% are made when determining the sperm concentration. Based on the concentration determination, dilution rates and number of semen doses are finalized. As ca be seen, at error rates within the range estimated, less than optimal dilution rates. It is clear from the literature that AI with 2 to 10 billion sperm can produce optimal fertility results in females when used within 2 days. However, what is not clear is the effect of the different concentrations and interaction with sperm storage, and how this might effect semen fertility and fertility in breeding herds. From the data it is clear that under estimation of sperm numbers leads to excessive sperm concentration and reduced value due to lower numbers of doses obtained. In the case of overestimation of sperm concentration, the optimal sperm numbers are not obtained, and with storage over the next 3-4 days, fertility level in the extended sperm is expected to decline. What is not clear is how the reduced fertility will ultimately impact the breeding herds that utilize multiple doses to compensate for the expected spread in time from insemination to fertilization. Regardless of the original ejaculate concentration (higher or lower than the example of 250 million/ml), the errors in final sperm concentration are similar. Scenario Error Conc.(x 10 6 ) Vol Dil SP(%) sperm.(x 10 9 ) doses vol(f) actual.(x 10 9 ) c-doses Cost/collect A -50% 125.0 200 1:3 29 25 8.3 667 6.0-8.2 -$41.00 B -25% 187.5 200 1:5 20 37 12.3 1,000 4.0-4.2 -$21.00 C 0 250.0 200 1:7 15 50 16.5 1,333 3.0 ----- ------ D +25% 310.0 200 1:8 12 62 20.6 1,653 2.4 + 4.1 +$20.50 E +50% 375.0 200 1:10 10 75 25.0 2,000 2.0 +8.5 +$42.50 Semen dose valued at $5.00/dose Desired 3 billion sperm /80 cc Error = Percentage error in estimated sperm concentration from actual Vol = volume of ejaculate Dil = final dilution SP(%)= final seminal plasma percentage Doses = number of doses to be produced vol(f) = final extension volume actual.(x 10 9 ) = sperm concentration in doses for final use c-doses = change in doses produced Cost/collect = cost relative to doses above or below optimal 31