J. Appl. Poult. Res.
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J APPL POULT RES 2006. 15:280-286
© 2006 Poultry Science Association
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Research Reports

Application of the Sperm Mobility Assay to Primary Broiler Breeder Stock

D. P. Froman1

Department of Animal Sciences, 112 Withycombe Hall, Oregon State University, Corvallis 97331

Correspondence: 1 Corresponding author: David.Froman{at}oregonstate.edu


   SUMMARY
TOP
 SUMMARY
DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
A new quantitative trait affecting male fertility was discovered in the 1990s. The term sperm mobility denotes the net movement of a sperm cell population against resistance at body temperature. Even though sperm cells are self-propelled DNA delivery vehicles, their self-propulsive nature is neither uniform among sperm within an ejaculate nor among males within flocks. Such variation is evident when semen quality is evaluated by sperm penetration of an Accudenz solution, hence the term sperm mobility assay. It is noteworthy that populations showing modest variation with respect to semen volume or sperm concentration often show considerable variation with respect to sperm mobility. Likewise, it is noteworthy that broiler breeder fertility is a function of sperm mobility phenotype when hens are inseminated artificially. This article outlines the following: 1) a series of experiments in which the sensitivity of the commercial sperm mobility assay was improved using semen donors from lines of chickens selected for either low or high sperm mobility and 2) the application of the improved technique to pedigree broiler breeders. Male fertility is subject to genetic selection when sperm mobility is the selection criterion. Therefore, sperm mobility may be a useful trait for improving broiler breeder reproductive efficiency.

Key Words: broiler breeder • chicken • fertility • semen quality • sperm


   DESCRIPTION OF PROBLEM
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Broiler breeder reproductive efficiency is affected by a variety of interacting variables. These include the combined effects of sexual behavior and social status, the effect of feeding regimens on the emergence and maintenance of a competent hypothalamo-hypophyseal-gonadal axis, and the impact of selection for growth. In regard to the last variable, it is axiomatic that reduced male fecundity is a necessary concomitant of genetic gain in growth [1]. Even though there is scientific and anecdotal evidence for this axiom, the scientific evidence is often based upon subjective measurements of sperm cell motility at ambient temperature. This is problematic because temperature has a profound effect upon sperm cell velocity [2], a critical variable affecting semen quality [3]. Whereas a variety of objective means of assessing poultry sperm motility have been described [3, 4], few of these techniques affect poultry reproductive management apart from the sperm quality index [5].

Application of the sperm mobility assay in a research context has resulted in 7 novel conceptual advances in the field of poultry reproduction. First, a new quantitative trait was discovered [6]. Second, fertility was a function of sperm mobility phenotype when hens were inseminated artificially [7]. Third, sperm mobility phenotype is determined by the size of the subpopulation of sperm within an ejaculate with a straight-line velocity (VSL) >30 µm/s [3, 8]. In other words, whereas every mobile sperm cell must be motile, not every motile sperm cell is mobile. Fourth, the mitochondrion was proven to be the critical organelle for phenotypic expression [9, 10]. Fifth, sperm residence within and emergence from the hen’s sperm storage tubules are most readily explicable in terms of sperm cell motility [2]. Sixth, sperm mobility phenotype is subject to genetic selection [9, 11]. Seventh, a model has been proposed linking phenotypic expression with sperm N-methyl-D-aspartate channels, seminal plasma glutamate, and formation of the mitochondrial permeability transition pore [12], a phenomenon associated with programmed and necrotic cellular death. Of these advances, the most salient was the demonstration that male fertility is subject to genetic selection when sperm mobility is the selection criterion.

The objective of the present work was to test whether the sperm mobility assay might be useful in an industry context as well. However, preliminary work was warranted because the commercial version of the sperm mobility assay contained glucose. This formulation represented the state-of-the-art of the 1990s [13]. In contrast, recent research [2, 8, 9, 10, 12] has identified key control points and pathways that enable sperm cell motility under physiological conditions that do not include glucose. Therefore, the present work outlines experiments designed to enhance the sensitivity of the commercial sperm mobility assay and describes the application of the improved assay using pedigree broiler breeders.


   MATERIALS AND METHODS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Sperm mobility assay reagents and equipment in these experiments were provided by Animal Reproduction Systems [14]. The assay was performed as outlined in References and Notes [15]. The first preliminary experiment was performed to illustrate 2 working principals of the assay. Two sperm suspensions were prepared per ejaculate. High sperm mobility New Hampshire roosters from the author’s line were used as semen donors (n = 12). One suspension was heated at 56°C for 5 min while the other was kept at room temperature (control sperm). After the sperm mobility index was measured for control sperm, each cuvette was covered and inverted, and a second measurement was taken to determine the maximal measurement possible. Thereafter, a second assay was performed with sperm rendered immotile by heating. Data were analyzed with a randomized complete block design [16].

A second preliminary experiment was performed to test the effect of glucose on the magnitude of sperm mobility measurements. In this case, roosters from the author’s line of low sperm mobility New Hampshires were used as semen donors (n = 18). Glucose-free experimental media (mobility buffer and 6%, wt/vol, Accudenz) were prepared as outlined in References and Notes [17]. Duplicate measurements were made per medium for each semen sample. Therefore, data were analyzed by nested AN-OVA [18]. Replicate observations per male were averaged, and averages were used to compute treatment group means.

A third preliminary experiment was performed to illustrate the relationship between the shape of a VSL frequency distribution and sperm mobility phenotype. Computer-assisted sperm motion analysis and construction of distributions were performed as outlined by Fro-man [2]. A single representative semen donor was selected from each line of New Hampshire roosters. Sperm mobility was confirmed [15] using glucose-free reagents [17] prior to computer-assisted sperm motion analysis.

Based upon the outcome of the second preliminary experiment, glucose-free reagents were custom-made by Animal Reproduction Systems [14]. These reagents were used to evaluate sperm mobility phenotype in pedigree broiler breeders. Two hundred forty-eight individual roosters were evaluated over a 4-wk interval. Roosters ranged from 29 to 56 wk of age. A single measurement was made in most cases. However, repeated measurements (n = 4 per male) were made for 65 roosters ranging in age from 35 to 45 wk of age.

The overall project entailed 4 objectives. The first objective was to evaluate phenotype as a function of time. Groups of roosters within a single line varying in age were used for this purpose due to time constraints. Data were plotted as a function of time using approximately 20 replicate observations per time point. Parameters of the function y(x) = {alpha}+ßx were estimated by the method of least squares [19] because raw data approximated a linear relationship. The second objective was to compare variation within and among roosters. This was performed with replicate measurements and nested ANOVA [18]. The third objective was to conduct correlation analyses [20]. The sperm mobility index was correlated against sperm concentration (n = 559 data pairs). Likewise, the sperm mobility index was correlated against body weight at 6 wk of age (n = 248 data pairs). The fourth objective was to construct a frequency distribution for sperm mobility phenotype. A male’s score was based on an average when multiple measurements were available. Individual scores were assigned to frequencies on the basis of increments of 5 sperm mobility index units (e.g., 0 to 4 and 5 to 9). The Kolmogorov-Smirnov test for goodness of fit [21] was used to determine whether observed frequencies approximated a normal distribution.


   RESULTS AND DISCUSSION
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Figure 1Go illustrates 2 key principles of the sperm mobility assay. First, as evidenced by application of heat-denatured sperm, penetration of the Accudenz layer was negligible in the case of immotile sperm, for which the mean sperm mobility index ±standard deviation was 1 ±0.6 units. In contrast, the majority of intact sperm entered the Accudenz layer within 5 min when the assay was performed at 41°C, the body temperature of the fowl. Mixing the sperm suspension overlay with the Accudenz layer maximized the number of sperm in the light path of the ARS 596A Mobility Analyzer (Figure 1Go). Thus, mixing immobile with mobile sperm yielded an upper limit. The mean ±standard deviation for mixed cuvettes was 87 ±2.4, which was equivalent to the maximum instrument value of 90 units. Thus, there was good correspondence between the total number of sperm used in the assay and instrument capability. It is noteworthy that this number of sperm cells is comparable to the number of sperm typically used to artificially inseminate a single hen. More importantly, the intraassay coefficient of variation was 3%, which denotes acceptable precision with respect to combined errors arising from sperm concentration estimation and pipetting.


Figure 1
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  		Figure 1. Sperm mobility index as determined by 3 conditions [28]. Mixed denotes the combination of the overlay and the Accudenz layer after completion of the assay at body temperature. Thus, this value represents the summation of mobile and immobile sperm within a test sample (i.e., the value that would be obtained if all sperm were mobile).High sperm mobility roosters served as semen donors. Each bar denotes a mean ±standard deviation (n = 12).

 
Once assay utility was confirmed with respect to sperm numbers, the next step was to evaluate the extent to which glucose might affect the magnitude of the sperm mobility index. Glucose had little effect when high sperm mobility males were used as semen donors (data not shown). In contrast, glucose affected the magnitude of the sperm mobility index (P < 0.0001) when low sperm mobility males were used (Table 1Go). Specifically, glucose increased the sperm mobility index 1.7-fold. Treatment means ±SEM were 27 ±3.3 and 16 ±1.5 units for media with and without glucose, respectively. As shown in Table 1Go, this effect was uniform among roosters (P > 0.05). Therefore, it was concluded that deletion of glucose from assay media would increase the range of observed values and thereby increase the sensitivity of the assay.


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  		Table 1. Summary of nested ANOVA testing the effect of deleting glucose from media used for the sperm mobility assay1
 
Computer-assisted sperm motion analysis was used to illustrate the relationship between VSL and sperm mobility phenotype. Previous research [8] has demonstrated that motile concentration and VSL are critical variables when explaining sperm mobility in terms of the motion of individual sperm cells. However, correlation coefficients ≤0.7 were observed when sperm mobility was correlated against motile concentration or average VSL. A breakthrough occurred when a linear relationship was demonstrated between the size of a sperm cell sub-population within an ejaculate characterized with a VSL >30µm/s and sperm mobility [3]. The size of this subpopulation can be estimated when the VSL of individual sperm are expressed as a frequency distribution (Figure 2Go). This concept is critical for understanding the value of the sperm mobility assay because the assay estimates the tendency of a male’s sperm to function effectively as self-propelled DNA delivery vehicles.


Figure 2
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  		Figure 2. Straight-line velocity (VSL) distributions for representative low and high sperm mobility roosters. Distributions were constructed from samples of 854 and 835 replicate sperm cells, and sperm mobility index values were 24 and 83 for low and high mobility males, respectively. Sperm mobility phenotype is proportional to the size of a distribution’s upper tail (i.e., the number of high velocity sperm in a semen sample).

 
The discovery of extreme phenotypic variation among males in previous work [6, 7, 8] prompted several critical questions. Of these, the most important was whether phenotype would be stable over the course of time. This question stemmed from the fact that most reproductive traits are dynamic (e.g., semen production or hen-day egg production). To date, time course studies have shown that low and high mobility phenotypes remain distinct in chickens [6, 22, 23]. Long-term repeated measurement was not possible in the present work. However, availability of males representing the span of semen production afforded an opportunity for analyzing sperm mobility phenotype through time within a line using a combination of single and repeated measurements per male (Figure 3Go).


Figure 3
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  		Figure 3. Sperm mobility index of pedigree broiler breeder males plotted as a function of time. Approximately 20 males were evaluated each week. Repeated symbols denote repeated measure of the same set of males. The solid line denotes the predicted function y(x) = {alpha} + ß(x). The slope did not differ from zero (P > 0.05).

 
Whereas the predicted curve in Figure 3Go demonstrated that flock phenotype was independent of time (P > 0.05), it could not address the question of variability among males within a flock. Consequently, a nested ANOVA was performed to provide assurance that variation observed on any given day was due to variation among males as opposed to random variation among observations. As expected, highly significant (P ≤0.0001) variation was observed among males (Table 2Go). These experimental outcomes (Figure 3Go and Table 2Go) afforded reasonable assurance that observations could be pooled to construct the first known frequency distribution depicting the range and central tendency of sperm mobility phenotype in pedigree broiler breeders (Figure 4Go). Even though data approximated a normal distribution (P > 0.05), the central tendency was within the same range as the low sperm mobility phenotype (Figure 5Go) found within a population of randomly bred New Hampshire roosters [24]. Finally, neither body weight at 6 wk of age (r = 0.04) nor sperm concentration (r = 0.15) was highly correlated with sperm mobility index.


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  		Table 2. Summary of nested ANOVA testing for variation in sperm mobility among males within a flock1
 

Figure 4
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  		Figure 4. Frequency distribution derived from categorization of sperm mobility data obtained from each of 248 pedigree line broiler breeder roosters. Sperm mobility was measured as an index ranging from 0 to 90 units. Observations were assigned to categories based on increments of 5 sperm mobility index units (e.g., 0 to 4 units).

 

Figure 5
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  		Figure 5. Comparison of sperm mobility frequency distributions between a line of pedigree broiler breeders (population 1; n = 248) and random bred New Hampshire roosters (population 2; n = 509). Data were normalized as a percentage of the maximal observed frequency within a distribution due to the difference in population sizes. Observations were assigned to categories based on increments of 5 sperm mobility index units.

 
In summary, the commercial sperm mobility assay was improved for use with broiler breeders. Deletion of glucose improved assay sensitivity by increasing the range of potential observations. Historically, the inclusion of glucose in semen diluents stemmed from an interest in semen preservation [25] rather than mechanisms that enable sperm motility in vivo. In contrast, recent investigations have demonstrated that fowl sperm use Ca2+ cycling [10] to activate phospholipase A2, an enzyme that hydrolyzes membrane phospholipids, presumably within the outer mitochondrial membrane. Activated phospholipase A2 generates palmitic and stearic acid for ß-oxidation within the mitochondrial matrix [2]. Thus, fowl sperm use an endogenous energy reserve to move against resistance over the hen’s vaginal surface. Consequently, use of glucose-free media afforded a more realistic estimate of sperm cell mobility.


   CONCLUSIONS AND APPLICATIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES
 

  1. Whereas glucose can improve sperm mobility in vitro, this does not mean that addition of glucose to a semen diluent will improve sperm mobility in vivo. The inclusion of glucose in diluents can prolong the potential of artificially preserved sperm to be motile following artificial insemination.
  2. A sperm mobility measurement reflects a male’s effective insemination dose. The traditional definition of insemination dose is the number of sperm cells inseminated per hen. However, immobile sperm cells, although viable, compromise the effectiveness of any given insemination dose. This is why broiler breeder fertility was a function of sperm mobility in previous work.
  3. Unlike many reproductive attributes, sperm mobility phenotype appears to be independent of time. Spermatogenesis is affected by dynamic hormone concentrations and receptor densities within the hypothalamus, adenohypophysis, and testis. In contrast, sperm mobility phenotype is most likely determined by an interaction between sperm cells and seminal plasma. This interaction always results in the formation of the mitochondrial permeability transition pore in some sperm within an ejaculate. Thus, the difference between low and high sperm mobility phenotypes is the extent to which this happens among sperm within an ejaculate. Mitochondrial function is pivotal to phenotypic expression, and mitochondria contain approximately 1,000 distinct proteins [26]. The opening of the mitochondrial permeability transition pore is controlled by a variety of proteins and energetic conditions [27]. Consequently, there may be common outcome for different reasons. It is proposed that variation in sperm mobility phenotype is a contributing factor in differences in male fertility among breeds of chickens.
  4. Sperm mobility phenotype is subject to genetic selection. There was no correlation between body weight at 6 wk of age and sperm mobility in the present work. Furthermore, appreciable variation in sperm mobility phenotype was observed among pedigree males as evidenced by a coefficient of variation of 52%. Thus, selection for sperm mobility may have potential for improving male reproductive efficiency without compromising body weight.


   ACKNOWLEDGMENTS
 
Powdered Accudenz was a gift from Accurate Chemical and Scientific Corporation (Westbury, NY).


   REFERENCES AND NOTES
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 

  1. Chambers, J. R. 1990. Genetics of growth and meat production in chickens. Pages 599–643 in Poultry Breeding and Genetics. R. D. Crawford, ed., Elsevier, Amsterdam, The Netherlands.
  2. Froman, D. P. 2003. Deduction of a model for sperm storage in the oviduct of the domestic fowl (Gallus domesticus). Biol. Reprod. 69:248–253.[Abstract/Free Full Text]
  3. Froman, D. P., E. R. Bowling, and J. L. Wilson. 2003. Sperm mobility phenotype not determined by sperm quality index. Poult. Sci. 82:496–502.[Abstract/Free Full Text]
  4. Froman, D. P., and D. J. McLean. 1996. Objective measurement of sperm motility based upon sperm penetration of Accudenz. Poult. Sci. 75:776–784.[ISI][Medline]
  5. Parker, H. M., and C. D. McDaniel. 2002. Selection of young broiler breeders for semen quality improves hatchability in an industry field trial. J. Appl. Poult. Res. 11:250–259.[Abstract/Free Full Text]
  6. Froman, D. P., and A. J. Feltmann. 1998. Sperm mobility: A quantitative trait of the domestic fowl (Gallus domesticus). Biol. Reprod. 58:379–384.[Abstract/Free Full Text]
  7. Froman, D. P., A. J. Feltmann, M. L. Rhoads, and J. D. Kirby. 1999. Sperm mobility: A primary deterimant of fertility in the domestic fowl (Gallus domesticus). Biol. Reprod. 61:400–405.[Abstract/Free Full Text]
  8. Froman, D. P., and A. J. Feltmann. 2000. Sperm mobility: Phenotype in roosters (Gallus domesticus) determined by concentration of motile sperm and straight line velocity. Biol. Reprod. 62:303–309.[Abstract/Free Full Text]
  9. Froman, D. P., and J. D. Kirby. 2005. Sperm mobility: Phenotype in roosters (Gallus domesticus) determined by mitochondrial function. Biol. Reprod. 72:562–567.[Abstract/Free Full Text]
  10. Froman, D. P., and A. J. Feltmann. 2005. Fowl (Gallus domesticus) sperm motility depends upon mitochondrial calcium cycling driven by extracellular sodium. Biol. Reprod. 72:97–101.[Abstract/Free Full Text]
  11. Froman, D. P., T. Pizzari, A. J. Feltmann, H. Castillo-Juarez, and T. R. Birkhead. 2002. Sperm mobility: Mechanisms of fertilizing efficiency, genetic variation and phenotypic relationship with male status in the domestic fowl, Gallus gallus domesticus. Proc. R. Soc. Lond. B Biol. Sci. 269:607–612.[Medline]
  12. Froman, D. P., J. C. Wardell, and A. J. Feltmann. 2005. Sperm mobility: Deduction of a model explaining phenotypic variation in roosters (Gallus domesticus). Biol. Reprod. 74:487–491.
  13. Ashizawa, K., G. J. Wishart, and Y. Tsuzuki. 2000. Avian sperm motility: Environmental and intracellular regulation. Avian Poult. Biol. Rev. 11:161–172.
  14. Animal Reproduction Systems, Chino, CA.
  15. The Animal Reproduction Systems (ARS) sperm mobility assay was performed as follows. Unless noted, reagents and equipment were provided by ARS. First, 2 anodized metal cuvette warming blocks were set upon a slide warmer (ARS Model 581A MOD 1) set at 41°C. Sealed, standard polystyrene cuvettes containing 6% (wt/vol) Accudenz were prewarmed within the warming blocks. Each cuvette contained 3 mL. In addition, 2 standard polystyrene cuvettes were set in a warming block. Each was filled with approximately 3 mL of mobility buffer and covered with a plastic cap. Reagent temperature was confirmed with a Type-K beaded thermocouple connected to a digital hand-held thermometer (VWR International, http://www.vwrsp.com). A 100-µL subsample of an ejaculate was placed in a 12 x 75 mm polystyrene test tube (VWR International). An 11.4-µL volume of semen was removed with a positive displacement pipet and added to 3.42 mL of 3% (wt/vol) NaCl dispensed into a standard polystyrene cuvette. The cuvette was covered with a plastic cap, and sperm was mixed by cyclic inversion of the cuvette. Sperm concentration was determined with an ARS 596A Mobility Analyzer, which measures sperm concentration, calculates buffer volume required for the assay, and measures sperm mobility as an index. After the foil tab was removed from a cuvette, the calculated volume of prewarmed buffer (e.g., 0.385 mL corresponding to an observed sperm concentration of 4.35 x 109/mL) was pipetted into an empty 12 x 75 polystyrene test tube. A 50-µL volume of semen was added with a positive-displacement pipet, and the contents were mixed by gentle vortexing. Thus, a sperm suspension was produced that contained 5 x 108 sperm/mL. A 300-µL volume of sperm suspension was immediately overlaid on the Accudenz solution. The cuvette was gently removed from the warming block after 5 min of incubation and then transferred to the well of the ARS 596A Mobility Analyzer to measure sperm mobility.
  16. Sokal, R. R., and F. J. Rohlf. 1969. Two-way analysis of variance. Pages 299–342 in Biometry. W. H. Freeman and Co., San Francisco.
  17. The modified mobility buffer was 50 mM N-Tris-[hydroxy-methyl]methyl-2-amino-ethanesulfonic acid (TES; Sigma Chemical Co., St. Louis, MO), pH 7.4, containing 128 mM NaCl and 2 mM CaCl2 (TES-buffered saline). The TES-buffered saline was used for preparing sperm suspensions and a 6% (wt/vol) Accudenz solution.
  18. Sokal, R. R., and F. J. Rohlf. 1969. Nested Analysis of variance. Pages 253–298 in Biometry. W. H. Freeman and Co., San Francisco.
  19. Sokal, R. R., and F. J. Rohlf. 1969. Regression. Pages 404–493 in Biometry. W. H. Freeman and Co., San Francisco.
  20. Sokal, R. R., and F. J. Rohlf. 1969. Correlation. Pages 494–548 in Biometry. W. H. Freeman and Co., San Francisco.
  21. Sokal, R. R., and F. J. Rohlf. 1969. Analysis of frequencies. Pages 549–620 in Biometry. W. H. Freeman and Co., San Francisco.
  22. Froman, D. P., A. J. Feltmann, and D. J. McLean. 1997. Increased fecundity resulting from semen donor selection based upon in vitro sperm motility. Poult. Sci. 76:73–77.[Abstract/Free Full Text]
  23. Bowling, E. R., D. P. Froman, A. J. Davis, and J. L. Wilson. 2003. Attributes of broiler breeder males characterized by low and high sperm mobility. Poult. Sci. 82:1796–1801.[Abstract/Free Full Text]
  24. Froman, D. P., 2005, unpublished data.
  25. Wishart, G. J. 1982. Maintenance of ATP concentrations in and of fertilizing ability of fowl and turkey spermatozoa in vitro. J. Reprod. Fertil. 66:457–462.[Abstract/Free Full Text]
  26. Scheffler, I. E. 1999. Nuclear genes encoding mitochondrial proteins. Pages 68–81 in Mitochondria. Wiley-Liss, San Diego.
  27. Nicholls, D. G., and S. J. Ferguson. 2002. Mitochondria in the cell. Pages 249–270 in Bioenergetics 3. Academic Press, San Diego.
  28. The sperm mobility assay entailed overlaying a sperm cell suspension on a solution of 6% (wt/vol) Accudenz in a cuvette, incubating the cuvette for 5 min at body temperature, and then measuring the extent to which sperm have penetrated the underlying Accudenz layer. Rendering sperm immotile by heat denaturation prior to overlay resulted in a negligible sperm mobility index because immotile sperm are immobile. In contrast, mobile sperm readily penetrated the Accudenz solution.




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