H. M. Parker and C. D. McDaniel 1. Poultry Science Department, Mississippi State University, Mississippi State, Mississippi 39762

Transcription

1 Semen Dilution Prior to Analysis Influences the Ability of the Sperm Quality Analyzer to Predict Fertility Whether Inseminating With a Constant Number of Sperm or a Constant Volume of Semen 1,2 H. M. Parker and C. D. McDaniel 1 Poultry Science Department, Mississippi State University, Mississippi State, Mississippi ABSTRACT Previous research has shown that the determine which semen dilution rate yielded an SQI that sperm quality index (SQI) is positively correlated with semen characteristics as well as fertility when broiler was most predictive of fertility, Pearson s correlation coefficients were obtained between the SQI at each dilution breeder semen is diluted 10-fold prior to analysis. However, research has not been conducted to establish if se- to diluent. Sperm quality index values declined logarith- rate and fertility. There was no difference in the SQI due men dilution rate affects the ability of the SQI to predict mically as live sperm concentration decreased. The SQI fertility when inseminating a constant number of sperm was most sensitive to live sperm concentrations from 11 or a constant volume of semen. As a result, 3 experiments to sperm/ml. The SQI for semen diluted 10- were undertaken. The objective of the first study was to fold was the best predictor of fertility when inseminating determine if diluent, minimum essential media, or 0.85% with a constant volume of 4-fold diluted semen (r = 0.71). saline affects the SQI. The second objective was to identify When hens were inseminated with a constant potential semen dilution rates for the SQI by obtaining the range in live sperm concentrations to which the SQI sperm/hen, the SQI for semen diluted 10- and 25-fold as is most sensitive. The purpose of experiment 3 was to well as to a constant concentration of sperm/ determine which semen dilution rate, 10-, 25-, 50-, 75- ml was equally effective at predicting fertility (r = 0.59, fold, or a constant concentration of sperm/ml, 0.52, and 0.61, respectively). Apparently, the SQI is predictive of fertility when semen samples are diluted 10- yields an SQI that is most predictive of fertility. Once a week for 3 wk, 20 hens were individually inseminated with either 20 µl of 4-fold diluted semen or a constant sperm from each of 29 broiler breeder males. To fold prior to analysis regardless of insemination method used. Dilution beyond 10-fold appears to alter semen quality, rendering the SQI less accurate. (Key words: artificial insemination, broiler breeders, fertility, sperm quality index) 2003 Poultry Science 82: INTRODUCTION Research has shown that the sperm quality index (SQI) is correlated with semen characteristics in humans (Bartoov et al., 1991; Matilsky et al., 1993; Johnston et al., 1995; Shibahara et al., 1997), bulls (Zavos et al., 1996; Cent et al., 2002), dogs (Iguer-Ouada and Verstegen, 2001), and poultry (McDaniel et al., 1998; Neuman et al., 2002a). The SQI is generated by measuring the number of disruptions in a light path created by sperm movement within a capillary. In poultry, McDaniel et al. (1998) revealed that 2003 Poultry Science Association, Inc. Received for publication June 2, Accepted for publication July 24, This is Journal Article Number J10362 from the Mississippi Agricultural and Forestry Experiment Station supported by MIS Use of trade names in this publication does not imply endorsement by the Mississippi Agricultural and Forestry Experiment Station of the products, nor similar ones not mentioned. 3 To whom correspondence and reprint request should be addressed: cmcdaniel@poultry.msstate.edu. the SQI is influenced by sperm concentration, viability, and motility and is indicative of overall semen quality in broiler breeders. The correlation of the SQI to semen quality in humans and dogs was obtained with undiluted semen samples (Bartoov et al., 1991; Matilsky et al., 1993; Johnston et al., 1995; Shibahara et al., 1997; Iguer-Ouada and Verstegen, 2001). However, due to high sperm concentrations and extremely low SQI readings in undiluted poultry semen, McDaniel et al. (1998) indicated that the SQI may be more predictive of rooster semen quality when semen is diluted at a constant rate prior to analysis. McDaniel et al. (1998) suggested a semen dilution rate of 10-fold prior to SQI analysis, because semen samples diluted less than 5-fold did not yield maximal SQI readings. Neuman et al. (2002b) proposed a 50-fold semen dilution for turkeys because tom sperm concentration is higher than that of Abbreviation Key: MEM = minimum essential medium; SQI = sperm quality index. 1808

2 SEMEN DILUTION FOR SPERM QUALITY INDEX 1809 the rooster. However, the exact effect of rate of semen dilution prior to SQI analysis on ability of the SQI to predict semen quality or fertility has not been ascertained. Parker et al. (2000) revealed a significant correlation of the SQI with live sperm concentration when broiler breeder semen was diluted 10-fold prior to analysis for the SQI. This relationship between the SQI and live sperm concentration was stronger than that of total sperm concentration or sperm viabililty alone with the SQI. Again, however, a semen dilution rate other than 10-fold may yield an even stronger relationship between the SQI and semen quality. An evaluation of the relationship for the SQI over its entire range with live sperm concentration may provide potential semen dilution rates that would yield an SQI that is the most indicative of semen quality and fertility. For example, if the relationship between the SQI and live sperm concentration is curvilinear, then the SQI may be most sensitive to live sperm concentration over a narrow range. In addition to semen characteristics, the SQI is also representative of fertility in humans (Matilsky et al., 1993; Shibahara et al., 1997) and poultry (McDaniel et al., 1998; Parker et al., 2000, 2002) as well as hatchability in poultry (Parker and McDaniel, 2002). Parker et al. (2000, 2002) found that selecting broiler breeder males for the SQI using a 10-fold semen dilution, improved fertility. In a field trial, Parker and McDaniel (2002) selected males for house placement based on either physical appearance or the SQI using a 10-fold semen dilution rate. Hatchability for males selected for the SQI was 1.1% higher than that of males selected for house placement based on physical appearance. However, again, it is possible that a 10-fold semen dilution may not yield an SQI that is the best predictor of fertility. Furthermore, insemination method may influence the ability of the SQI to predict fertility. As opposed to insemination with a constant number of sperm, insemination of hens with a constant volume of semen or natural mating allows for variation in sperm concentration among males to influence fertility (Wilson et al., 1979). Because sperm concentration influences the SQI, insemination of hens with a constant number of sperm may restrict the ability of the SQI to predict fertility. Additionally, composition of the semen diluent may also influence the SQI. McDaniel et al. (1998) used a more complete nutritive diluent, minimum essential media, to determine the SQI, yet research involving avian fertility and hatchability utilized 0.85% saline (Parker et al., 2000, 2002; Parker and McDaniel, 2002). Therefore, 3 experiments were undertaken. In experiment 1, the objective was to determine the effects of 2 different diluents on the SQI. In experiment 2, the objective was to identify potential semen dilution rates for the SQI by obtaining the range in live sperm concentration to which the SQI is most sensitive. The objective of experi- 4 Medical Electronic Systems Ltd., Migdal Haemek, Israel. ment 3 was to determine which semen dilution rate yields an SQI that is most predictive of semen quality and fertility for broiler breeder males. For experiment 3, hens were artificially inseminated with either a constant volume of semen or a constant number of sperm from each male to determine if the predictive ability of the SQI is dependent upon insemination method. MATERIALS AND METHODS Housing and Environment Twenty-nine Cobb broiler breeder males, 26 wk of age, were obtained from a local integrator. Males were housed in individual cages and maintained at 24 C. Broiler breeder males were fed the Mississippi State University male breeder diet (3,080 kcal ME/kg, 13.9% CP, and 1% Ca) and were feed-restricted according to the primary breeder s recommendations. All males received 16 h of light per d. Additionally, 30 Ideal 236 White Leghorn males, 44 wk of age, and 580 Hyline 36 Leghorn hens, 45 wk of age, were housed in individual cages. Leghorn hens and roosters consumed the Mississippi State University layer diet (2,860 kcal ME/kg, 14.5% CP, and 4% Ca) ad libitum and were exposed to light from 0500 to 2100 h. The Leghorn hens and roosters were caged in a house with conventional environmental controls. Experiment 1 Ejaculates from the Leghorn males were collected and pooled utilizing the method of Burrows and Quinn (1937). The semen pool was diluted with either 0.85% saline or minimum essential medium (MEM, Howarth, 1981) to obtain dilution factors of 10-, 25-, 50-, and 75-fold. The SQI was obtained simultaneously on three Sperm Quality Analyzers 4 for each SQI dilution. The experiment was replicated in triplicate. A new semen dilution tube was made for each SQI replicate, and the testing order was random. An assembly line was established so that every semen sample was analyzed within 1 min of dilution. Experiment 2 Ejaculates from Cobb males were collected and pooled. Sperm concentration of the semen pool was determined by using a hemacytometer. To determine sperm viability, 3 fluorometric (Bilgili and Renden, 1984) measurements were taken prior to dilution. Semen was diluted with 0.85% saline to yield live sperm concentrations of 12, 29, 58, 87, 116, 145, 174, 231, 463, 694, 926, and 1, sperm/ml. Three SQI readings were obtained for each semen dilution. A new semen dilution tube was made for each SQI replicate and the testing order was random. An assembly line was established so that every semen sample was analyzed within 1 min of dilution. Experiment 3 Ejaculates were collected weekly from 29 individual Cobb males for 3 wk. Each sample was diluted with 0.85%

3 1810 PARKER AND MCDANIEL TABLE 1. Effect of semen diluent on the sperm quality index (SQI) at each semen dilution rate in Experiment 1 Diluent Semen dilution rate Saline SQI MEM 1 SQI 10-fold 488 a 483 a 25-fold 308 b 299 b 50-fold 136 c 157 c 75-fold 65 d 77 d a d SQI means within a row and column with different superscripts are significantly different (n = 9, P < 0.05). 1 Minimum essential medium. saline to 10-, 25-, 50-, and 75-fold. Additionally, each sample was diluted to yield a constant concentration of 180 x10 6 sperm/ml. The sperm/ml concentration was selected because it was near the midpoint of the SQI curve generated in experiment 2. Each male s SQI was determined in duplicate, and each SQI was measured simultaneously on two Sperm Quality Analyzers. The CV between the two analyzers was 3.6%. An assembly line was established so that every semen sample was analyzed within 2 to 3 min of collection. For each semen sample, two fluorometric readings (Bilgili and Renden, 1984) were obtained on undiluted neat semen to determine sperm concentration and viability. After semen collection, dilution, and testing, two hen groups (10 hens/group) per male were inseminated with either a constant number of sperm or a constant volume of semen. Hens were inseminated within 5 min of semen collection. Semen utilized for artificial insemination was diluted with MEM. One group of hens per male were inseminated with 20 µl of diluted semen containing a constant concentration of sperm, and the other hen group received 20 µl of 4-fold diluted semen (1 part semen to 3 parts MEM). Eggs were collected, labeled, and stored daily (18.3 C), then incubated weekly. On d 6 of incubation, eggs were broken to determine the occurrence of embryonic development. Statistical Analyses Data from experiment 1 were analyzed as a generalized randomized complete block design with the three repli- cates as blocks. Means were separated using Fisher s protected least significant difference (P < 0.05). Curvilinear regression was used to reveal the relationship between live sperm concentration and the SQI of experiment 2. For the fertility data of experiment 3, semen treatments were applied to groups of hens, and groups of hens were the experimental units. Shapiro and Wilk (1965) coefficient (W) was used to test for normality of the SQI and semen characteristic populations. The distribution curves were considered normal as W approached 1 at P > For each SQI dilution rate, Pearson s correlation coefficients were obtained for the relationship of the SQI with semen characteristics and fertility. Correlation analyses were also conducted to examine the relationship of semen characteristics, other than the SQI, with fertility (Steel and Torrie, 1980). Correlation coefficients were separated (P < 0.05) using the method of Steiger (1980). Experiment 1 RESULTS There were no differences in SQI regardless of whether semen was diluted using 0.85% saline or MEM (Table 1). However, with each increasing dilution factor, the SQI declined. Experiment 2 The relationship of live sperm concentration to the SQI is represented in Figure 1. This graph reveals that SQI values increased logarithmically as live sperm concentrations increased (r 2 = 0.96, P < ). The SQI increased approximately 350 units, 18 to 374, when live sperm concentrations increased from 11 to sperm/ml. As live sperm concentration increased from 463 to sperm/ml, SQI values plateaued, increasing only 39 units, from 449 to 488. Experiment 3 Total sperm concentration, percentage of viable sperm, and live sperm concentration averaged sperm/ ml, 90.4%, and sperm/ml, respectively (data TABLE 2. Population distribution characteristics for total sperm concentration, percentage of viable sperm, live sperm concentration, and the sperm quality index (SQI) at each semen dilution rate in experiment 3 1 Skewness W P-value 2 Distribution Total sperm concentration Normal Viable sperm (%) Right Live sperm concentration Right SQI from 10-fold dilution Right SQI from 25-fold dilution Normal SQI from sperm/ml Normal SQI from 50-fold dilution Normal SQI from 75-fold dilution Left 1 These data are from averages obtained from individual ejaculates of 29 males for each of the 3 wk of insemination. 2 P-value for Shapiro and Wilk coefficient (W).

4 SEMEN DILUTION FOR SPERM QUALITY INDEX 1811 FIGURE 1. Relationship of live sperm concentration to the sperm quality index (SQI) in experiment 2. Each point represents the mean of 3 replicates from one pool of semen. Semen from 29 males was pooled together and then diluted from to 1, sperm/ml. A logarithmic increase in SQI was noted with increasing live sperm concentration (y = 120 Ln(x) 314, r 2 = 0.96, P < ). not shown). The population distribution for semen characteristics revealed that total sperm concentration was normally distributed, yet the percentage of viable sperm and live sperm concentration population distributions were skewed to the right (Table 2). Population distributions for the SQI at each semen dilution are shown in Table 2. As the dilution rate increased, the SQI population shifted from the right, toward normal, then to the left. Like the percentage of viable sperm and live sperm concentration, the population was skewed to the right for the 10-fold SQI dilution. However, the constant sperm/ml, 25-fold, and 50-fold SQI populations were normally distributed. The 75-fold SQI population was skewed to the left. The relationship of the SQI at different dilution rates with total, viable, and live sperm concentration is represented in Table 3. There was a nonsignificant relationship between total sperm concentration and the sperm/ml SQI dilution rate. The relationship of total sperm concentration with the SQI at a 10-fold dilution produced the highest correlation coefficient (r = 0.59); however, the coefficients for 25- and 50-fold dilutions (r = 0.58 and r = 0.47, respectively) were not different from that of the 10-fold SQI dilution. The SQI from the 75- fold dilution was not strongly correlated to total sperm concentration (r = 0.39). The SQI from the 10-fold dilution rate and the constant concentration of sperm/ ml yielded higher correlations with the percentage of viable sperm than the 50- and 75-fold SQI dilutions, (r = 0.69 and r = 0.62 versus r = 0.43 and r = 0.39, respectively). The strongest relationship between SQI and live sperm concentration was achieved with the 10-, 25-, and 50-fold dilutions (r = 0.72, r = 0.71, and r = 0.62, respectively). The relationship of the SQI with live sperm concentration was weaker than that of the SQI with the percentage of viable sperm for the constant concentration of sperm/ml SQI dilution. Correlation of the SQI from the 10-fold semen dilution with total sperm concentration, percentage of viable sperm, and live sperm concentration is presented in Figure 2. Correlation coefficients of the SQI from each semen dilution rate with fertility for each insemination method are presented in Table 4. When hens were inseminated with a constant volume of 1:4 diluted semen, on average, TABLE 3. Correlation coefficients for the sperm quality index (SQI) at each semen dilution rate with total sperm concentration, percentage of viable sperm, and live sperm concentration in experiment 3 1 Sperm characteristic SQI at each Total sperm Live sperm semen dilution rate concentration Viable sperm (%) concentration r 10-fold 0.59 a 0.69 a 0.72 a 25-fold 0.58 a NS 0.71 a sperm/ml NS 0.62 a 0.46 c 50-fold 0.47 a 0.43 b 0.62 ab 75-fold 0.39 b 0.39 b 0.53 bc a c Correlation coefficients within a column with different superscripts are significantly different (P < 0.05). NS indicates a nonsignificant correlation coefficient. 1 These data are from averages obtained from individual ejaculates of 29 males for each of the 3 wk of insemination.

5 1812 PARKER AND MCDANIEL FIGURE 3. The relationship of the sperm quality index (SQI) from the 10-fold semen dilution in experiment 3 with fertility from hens inseminated with A. a constant 1:4 semen dilution or B sperm per hen for each of the 3 wk of insemination (20 hens/male). Each group of 10 hens laid on average 8.4 eggs. FIGURE 2. The relationship of the sperm quality index (SQI) from the 10-fold semen dilution in experiment 3 with A. total sperm concentration, B. percentage of viable sperm, and C. live sperm concentration. These data are averages obtained from individual ejaculates of 29 males for each of the 3 wk of insemination. approximately sperm per hen, the SQI from the 10-fold dilution (r = 0.71) was the best predictor of fertility. The SQI from the constant concentration of sperm/ml dilution did not predict fertility as well as the 10-fold dilution. The SQI from the 25-fold dilution predicted fertility as well as the SQI from the sperm/ml dilution and was a better predictor of fertility than the SQI from the 50- and 75-fold dilutions. Inseminating hens with a constant number of sperm, approximately sperm per hen, revealed that the SQI from semen dilutions of sperm/ml, 10- fold, and 25-fold were the best indicators of fertility (r = 0.61, r = 0.59, and r = 0.52, respectively). The correlation coefficient for the SQI from the sperm/ml dilution rate with fertility was slightly higher when inseminating with a constant number of sperm versus a constant volume of semen (r = 0.61 versus r = 0.54). Correlation of the SQI from the 10-fold semen dilution with fertility and each insemination method is graphically presented in Figure 3. Correlation coefficients for individual semen characteristics with fertility from each insemination method are presented in Table 5. When hens were inseminated with a constant number of sperm, a weak relationship between fertility and the percentage of viable sperm (r = 0.49) was detected; however, there was no relationship between total sperm concentration or live sperm concentration and fertility for this insemination method. Inseminating hens with a constant volume of semen revealed a weak relationship for the percentage of viable sperm and live sperm concentration with fertility (r = 0.45 and r = 0.40, respectively). DISCUSSION As shown in experiment 1, there were no differences between SQI readings when semen was diluted with either 0.85% saline or the more complete diluent, MEM. Because semen dilution and SQI analysis occurs within 1 min, apparently the diluent has no effect on the SQI. It is possible that a difference due to diluent would exist in SQI readings if semen samples were stored longer in diluent prior to analysis. The findings from experiment 2 are consistent with those of McDaniel et al. (1998) which indicated that SQI values plateaued when total sperm concentrations were greater than sperm/ml. Also, this relationship

6 SEMEN DILUTION FOR SPERM QUALITY INDEX 1813 TABLE 4. Correlation coefficients for the sperm quality index (SQI) from each semen dilution rate with fertility for each artificial insemination method in experiment 3 Artificial insemination method 1 SQI at each 4-fold 45 million semen dilution rate dilution sperm/hen r 10-fold 0.71 a 0.59 ab 25-fold 0.59 b 0.52 ab sperm/ml 0.54 bc 0.61 a 50-fold 0.47 cd 0.44 b 75-fold 0.38 d NS a d Correlation coefficients within a column with different superscripts are significantly different (P < 0.05). NS indicates a nonsignificant correlation coefficient. 1 For each of 29 males, 10 hens were inseminated with 20 µl ofa1:4 constant volume of diluted semen and 10 hens were inseminated with sperm per hen for each of the 3 wk of insemination (20 hens/ male). Each group of 10 hens laid on average 8.4 eggs. of live sperm concentration with the SQI is very similar to that reported for turkey toms (Neuman et al., 2002a). The relationship of total sperm concentration with the SQI in dog semen revealed that the SQI plateaued at greater than 200 units (Iguer-Ouada and Verstegen, 2001). Overall these data seem to indicate that the SQI is more sensitive to changes in broiler breeder semen quality when low live sperm concentrations are evaluated in the capillary. However, correlation data from experiment 3 revealed that the SQI is actually more of an indicator of semen quality and fertility when samples are evaluated at higher live sperm concentrations. In experiment 2, sperm/ml was very near the midpoint of the SQI curve for live sperm concentration. However, in experiment 3, the SQI from samples either diluted 50-fold, on average sperm/ml, or to a constant concentration of sperm/ml was not the best overall indicator of fertility or semen quality. Actually, the SQI from the 10-fold dilution was most indicative of fertility and overall semen quality. Sperm concentration in the 10-fold SQI dilution was, on average, sperm/ ml and well outside of the most sensitive area of the SQI-live sperm concentration curve from experiment 2. Therefore, it is possible that the most sensitive area of the SQI-live sperm concentration curve from experiment 2 is an inaccurate indicator of true semen quality and may instead be an artifact of dilution alone. The relationship of the SQI from the 10-fold dilution also yielded the highest numeric correlation coefficient with total sperm concentration, and as dilution increased this relationship with total sperm concentration weakened. Other research has also revealed a strong relationship for the SQI with total sperm concentration when semen samples were diluted 10-fold (McDaniel et al., 1998; Parker et al., 2000). In bulls, a strong correlation has been shown between the SQI and sperm concentration (r = 0.98) at different dilution rates (Cent et al., 2002). Iguer- Ouada and Verstegen (2001) also reported that, in dogs, the SQI was strongly correlated with total sperm concentration (r = 0.84). The correlation coefficient for the 10-fold SQI dilution with sperm viability is similar to the findings of Parker et al. (2000) when semen was diluted 10-fold. In the present study, the correlation coefficient of the SQI from the constant sperm/ml dilution with the percentage of viable sperm was found to be numerically lower. As with total sperm concentration and the percentage of viable sperm, the relationship of live sperm concentration with the SQI weakened as live sperm concentrations decreased with increasing dilution. Because the SQI monitors gross sperm movement, any change in live sperm concentration would alter sperm movement within the capillary and influence SQI readings. However, because sperm concentration was controlled in the constant sperm/ml SQI dilution and dead sperm are immotile, only the rate of sperm movement influenced the SQI readings for this dilution. Research has shown that the SQI is influenced by both broiler breeder and turkey sperm motility (McDaniel et al., 1998; Neuman et al., 2002a). Using computerized sperm analysis, Integrated Visual Optical System (IVOS), it was revealed that the SQI is highly correlated to the percentage of motile (r = 0.9), rapid (r = 0.7), and progressive spermatozoa (r = 0.8) in dogs (Iguer-Ouada and Verstegen, 2001) as well as overall motile spermatozoa (r = 0.76) in bulls (Cent et al., 2002). In experiment 3, as semen was diluted beyond 10-fold, the predictive ability of the SQI declined. It is possible that at higher dilutions sperm motility is altered by dilution alone, lowering the correlation of the SQI with undiluted motile sperm concentration. Negative effects on sperm motility have been reported when poultry semen is diluted (Austin and Natarajan, 1991). In bulls, it has been established that reduction of seminal plasma by dilution is harmful to spermatozoa (Garner et al., 2001). Dilution of fresh fowl semen has been shown to have harmful effects on spermatozoa. These effects are believed to be caused by an interaction of seminal plasma with the diluent (Blesbois and de Reviers, 1992). With boars, increasing the dilution rate revealed increases in the proportion of abnormal acrosomes (Bamba and Cran, 1988). Omprakash et al. (1992) reported that when fowl semen was diluted 1:3 there was an increase in the percentage of abnormal spermatozoa due to dilution. Using turkey semen, Sexton (1976) found that a 4-fold dilution decreased respiration. Other research has shown that oxidative metabolism was higher in diluted turkey semen (Monsi and Enos, 1992). In experiment 3, the greatest amount of seminal plasma and sperm numbers were present when the 10-fold SQI dilution was used, and the SQI from the 10-fold dilution was most predictive of semen quality. Apparently by using a 10-fold semen dilution the SQI is more indicative of true, undiluted semen quality. In experiment 3, sperm concentration had no relationship with fertility, regardless of insemination method. However, when hens were inseminated with a constant volume of semen, which allowed variation in sperm concentration among males to influence fertility, the SQI from

7 1814 PARKER AND MCDANIEL TABLE 5. Correlation coefficients for each individual semen characteristic with fertility from each artificial insemination method in experiment 3 Artificial insemination method 1 4-fold 45 million Semen characteristic dilution sperm/hen r Total sperm concentration NS NS Viable sperm (%) Live sperm concentration 0.40 NS 1 For each of 29 males, 10 hens were inseminated with 20 µl ofa1:4 constant volume of diluted semen and 10 hens were inseminated with sperm per hen for each of the 3 wk of insemination (20 hens/ male). Each group of 10 hens laid on average 8.4 eggs. NS indicates a nonsignificant correlation coefficient. a 10-fold dilution was the best indicator of fertility. The correlation coefficients of the SQI with fertility declined in response to increased semen dilution and hence decreased sperm concentration. Inseminating hens with a constant number of sperm revealed that the relationship of the SQI with fertility was not as strong as the relationship reported for hens inseminated with a constant volume of semen. As stated previously, this could be due to the fact that the SQI is correlated to sperm concentration (McDaniel et al., 1998; Parker et al., 2000; Iguer-Ouada and Verstegen, 2001; Cent et al., 2002), and controlling sperm concentration during insemination partially prohibits the predictive power of the SQI. However, research has shown that fertility is improved when hens are inseminated with either a constant volume of semen or a constant number of sperm when the SQI from a 10-fold dilution is used to select roosters (Parker et al., 2000, 2002). Of course, in a natural mating situation, a hen receives a volume of semen not a constant number of sperm. Research has also shown that hatchability for naturally mating birds was improved by selecting males for the SQI prior to house placement, when using a 10-fold SQI dilution (Parker and McDaniel, 2002). Wishart and Palmer (1986) reported that sperm concentration had a low correlation (r = 0.053) with fertility when hens were inseminated with a constant concentration of sperm. The hens used in experiment 3 were inseminated with low numbers of sperm to ensure that the sperm storage tubules were not maximized. Possibly as a result of the low number of spermatozoa inseminated, sperm concentration was not the primary determinant of fertility for either insemination method. This is evidenced by the nonsignificant relationship of total sperm concentration with fertility, regardless of insemination method used. Furthermore, the SQI was a much better predictor of fertility, regardless of insemination method, than any of the other semen characteristics measured in this study. As previously reported by Parker et al. (2000), the most likely reason the SQI is superior to traditional semen characteristics in predicting fertility is because the SQI is influenced by sperm concentration, viability, and motility collectively. The relationship between the SQI and fertility became weaker with increasing semen dilution and therefore decreasing sperm concentration. As previously mentioned, it has been suggested that sperm motility is altered when semen is diluted in solutions other than seminal fluid (Grunert et al., 1989; Austin and Natarajan, 1991). In experiment 3, it is possible that as semen was increasingly diluted, motility was altered, inhibiting the ability of the SQI to predict true undiluted semen quality and hence fertilizing potential of individual males. Also, research has suggested that semen viscosity influences sperm motility (Amann and Hammerstedt, 1980). The 10-fold SQI dilution contained more seminal fluid than the other semen dilutions used in this trial; as a result, this dilution would have been more viscous. In experiment 3, it is possible that sperm became more dispersed with dilution, resulting in SQI readings that were truly not indicative of semen quality in an undiluted or slightly diluted sample and therefore not predictive of fertility. It is also possible that the reason the SQI at a 10- fold dilution was the best predictor of fertility was because it could be more indicative of the viscosity of the vaginal fluids as opposed to the other SQI dilutions. In conclusion, it is apparent that the 10-fold dilution is the best dilution for the SQI to predict fertility and determine semen quality. It is very likely that dilution beyond 10-fold alters true semen quality and therefore impairs the ability of the SQI to accurately predict semen quality and fertility. REFERENCES Amann, R. P., and R. H. 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