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From the * Department of Animal Sciences,
University of Illinois at Urbana-Champaign, Urbana, Illinois; and the
Department of Dairy Science, Virginia
Polytechnic Institute and State University, Blacksburg, Virginia.
| Correspondence to: Dr David Miller, 328 Animal Sciences Laboratory, 1207 W Gregory Dr, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (e-mail: d-mille{at}uiuc.edu). |
| Received for publication November 14, 2001; accepted for publication March 20, 2002. |
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Abstract |
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Key words: Fertilization, oocyte, accessory, cattle, nonreturn, heterospermic
One approach to solving this problem is to use laboratory assays that test a multitude of traits. Some assays, such as successful in vitro fertilization, require that spermatozoa be motile, have normal morphology, have an adequate complement of zona pellucida and oocyte membrane receptors, be able to activate oocytes, etc. The relationship of in vitro fertilization rates to fertility has been variable, probably because of the normal variability between batches of oocytes, variation in semen handling, and inadequate internal controls. A second approach to solving this problem is to identify the traits that most often cause reduced fertility and make those part of the routine semen analysis. The hypothesis tested in this report was that binding to the oocyte's zona pellucida accounts for significant variation in fertility when assessing fertility by a nonreturn to estrus rate and a competitive index. We also used semen from bulls that differed in the number of accessory spermatozoa found on embryos and ova to compare in vivo zona binding with in vitro competitive zona binding.
When testing whether a trait is related to fertility, one should consider fertility traits as divided into 2 components. One component includes defects that can be compensated for by inseminating more spermatozoal, so-called compensable traits (Saacke et al, 2000). Examples of these are spermatozoal viability, morphology, or factors that hinder the interaction of spermatozoa with the oocyte. If an animal has defects in compensable traits, reduced fertility is only observed when a less than optimal number of spermatozoa are inseminated. An excess of spermatozoa is usually inseminated in cattle, so laboratory assays that measure compensable traits are seldom highly correlated with bovine fertility. When excessive spermatozoa are inseminated, only the traits that cannot be compensated for are related to fertility; these traits are referred to as uncompensable. Examples of these traits are chromatin aberrations or morphological defects that do not affect sperm—oocyte interaction but that impair fertilization or embryogenesis once initiated. Although non-return rate is usually not well related to compensable traits, relative fertility based on the insemination of semen from 2 different males in competition would be affected by compensable traits (Saacke et al, 2000).
The goal of the experiments in this report was to determine if the first step in spermatozoa—oocyte interaction, zona binding, was related to fertility. This is considered a compensable trait because spermatozoa with an oocyte-binding defect would not be able to interact and fertilize an oocyte. Other normal spermatozoa in the sample could compensate for sperm defective in zona binding and could fertilize the oocyte. Previous studies aimed at determining if oocyte binding was related to fertility, assessed by a 56-day nonreturn rate, gave conflicting results (Fazeli et al, 1997; Zhang et al, 1998). The discrepancy may be due to the inability to control the variation in oocyte quality and spermatozoal preparation. Herein, we control this source of variation by using an in vitro competitive assay. Spermatozoa from 2 males were differentially stained, mixed, and allowed to bind to oocytes (Miller et al, 1998). This provides an internal control because spermatozoa bind to the same oocytes in the same droplet. Many samples can then be ranked by a series of pairwise comparisons. This principle has been used successfully with in vivo experiments to determine relative fertility when semen from 2 males was mixed and inseminated—so-called heterospermic insemination experiments (Saacke et al, 1980; Dziuk, 1996).
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Materials and Methods |
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Preparation of Semen From Bulls Whose Fertility Was Determined by
Competitive Insemination
Cryopreserved bovine semen from 8 bulls (3 Hereford, 3 Angus, and 2
Simmental) was obtained from Clif Marshall at Select Sires (Plain City, Ohio).
This semen had been prepared and used for a previous study involving 9 bulls;
only 8 samples were still available
(Saacke et al, 1980). Two or 3
ejaculates were collected in succession and pooled, providing sufficient semen
doses for insemination and laboratory study. Semen was extended to 60 x
106 sperm/mL in egg yolk-citrate-glycerol, packaged in 0.5-mL
French straws (Instruments de Médecine Vétérinaire,
l'Aigle, France), and frozen to —196°C following the optimal
procedure as described (Robbins et al,
1976). After plunging straws into 35°C water for 1 minute,
equal numbers of spermatozoa from 2 bulls were mixed and reloaded into French
straws for insemination. On the basis of the number (proportions) of calves
sired competitively, a competitive index was derived for each of the 8 bulls
(Saacke et al, 1980). For the
in vitro competitive-binding assay, semen samples were thawed and capacitated
as described above with 2 exceptions. Only 1 straw of semen was thawed from
each male, and 1.5 x 106 spermatozoa were capacitated in 100
µL of dmTALP with heparin (Parrish et
al, 1988).
Semen Collection and Processing for Quantification of Accessory
Spermatozoa
Semen for this experiment was the same as that used in the study of Dalton
et al (2001). Three Holstein
bulls, ranging from 2 to 9 years of age, were selected on the basis of neat
semen characteristics equal to or greater than 70% morphologically normal
spermatozoa and 60% estimated progressive motility. Two ejaculates taken in
succession from each bull were pooled and extended to 50 x
106 sperm/mL with clarified egg yolk-citrate-glycerol and
cryopreserved in the same manner as the semen used in the heterospermic trial
(above). For each bull, spermatozoa accessibility to the ovum in vivo was
measured by quantifying accessory spermatozoa number in ova or embryos
recovered nonsurgically 6 days postinsemination as described
(Dalton et al, 2001). For the
competitive zona-binding assay, semen samples were thawed and prepared as
described above with the exception that 4.5 x 106 spermatozoa
were capacitated in 300 µL of dmTALP with heparin
(Parrish et al,
1988).
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Differential Staining of Spermatozoa
Two fluorescent lipophilic dyes, DiQ
(4-[4-(dihexadecylamino)styryl]-N-methylquinolinium iodide), an orange-red
fluorochrome, and DiOC16 (3,3'-dihexadecyloxacarbocyanine
pechlorate), a yellow-green fluorochrome (Molecular Probes, Eugene, Ore), were
prepared in 10-mM stock solutions. DiQ was dissolved in dimethyl sulfoxide and
DiOC16 in dimethylformamide. The stock solutions were stored in
aluminum foil at -20°C. Prior to use, a 12.5-µL aliquot was thawed,
sonicated for 10 seconds, and centrifuged at 15 000 x g for 1
minute, and the supernatant was used to stain sperm. After 3 hours of
capacitation, spermatozoa were incubated with 83 µM of either dye for 60
minutes at 39°C in dmTALP. The sperm suspensions were centrifuged for 30
seconds at 10 000 x g, the supernatant was removed, and the
spermatozoa were suspended in 0.3 mL of dmTALP. To estimate motility, 100
spermatozoa were examined with a microscope using differential interference
contrast optics.
Bovine Oocyte Collection and Preparation
Cumulus oophorus-free mature bovine oocytes were obtained from BoMed
(Madison, Wis). These oocytes had been aspirated from 2- to 6-mm antral
follicles and were processed in 114 mM NaCl, 3.2 mM KCl, 2 mM sodium
bicarbonate, 0.4 mM monosodium phosphate, 10 mM lactate, 2.0 mM
CaCl2, 0.5 mM MgCl2, 10 mM HEPES, and 100 IU/mL
penicillin. Oocytes were stripped of cumulus cells by vortexing and shipped to
Urbana, Ill, at 39°C by overnight mail. Oocytes were shipped in TC199
medium with Earle salts supplemented with bovine-luteinizing hormone,
follicle-stimulating hormone, 0.22 mM sodium pryuvate, 25 µg/mL gentamicin,
and 10% fetal calf serum. Upon arrival, they were washed in medium B (127 mM
NaCl, 5.3 mM KCl, and 18.2 mM HEPES, pH 7.2) and then were fixed in 1.5%
formaldehyde in medium B for 10 minutes. The fixed oocytes were washed through
5 droplets of medium B and were transferred to a droplet of dmTALP covered
with mineral oil. They were stored in this droplet at 5°C for 1-25 days
until use. Prior to use in competitive sperm-binding assays, oocytes were
washed and placed in 25-µL drops of dmTALP under oil in groups of 10
oocytes per drop and equilibrated to 39°C.
Competitive Spermatozoa—Oocyte-Binding Assay
Following spermatozoa capacitation and staining, samples from 2 males were
used for a competitive spermatozoa—oocyte-binding assay. An example of
competition assignment is diagramed in the Table. This design allows indirect
and direct comparisons of zona-binding ability. Each assay consisted of 2
competitions in which the fluorochromes used for each of the 2 spermatozoa
samples were switched to minimize an effect of the fluorochromes. An equal
number (from 1.25 x 105 to 3.75 x 105) of
spermatozoa from each male labeled with either fluorochrome were mixed and
added to triplicate 25-µL droplets of dmTALP containing 10 oocytes each
equilibrated to 39°C. Gametes were coincubated for 15 minutes at 39°C
to allow maximal binding. After incubation, spermatozoa-bound oocytes were
transferred to a wash droplet of dmTALP by mouth pipette and then to a
25-µL droplet of 4% paraformaldehyde in phosphate-buffered saline. These
washing conditions were sufficient to remove any loosely adherent spermatozoa
on the oocyte. After the oocytes were washed, they were transferred to a
25-µL droplet of dmTALP on a microscope slide. The coverslip was prepared
by placing a small amount of petroleum jelly on each corner, to avoid crushing
the oocytes, and was placed gently over the droplet. The coverslip was sealed
with nail polish and stored in aluminum foil at room temperature until
counting. Spermatozoa were counted using a Zeiss Axioskop with fluorescence
optics (Zeiss, Thornwood, NY) within 24 hours of preparation. To detect
DiOC16 fluorescence, the Zeiss 09 filter set was used that, for
excitation, has a band-pass cutoff of 450-490 nm, a 510-nm beam splitter, and
a long-pass 515-nm emission filter. For detecting DiQ fluorescence, the Zeiss
15 filter set was used that has a band pass of 534-558 nm, a 580-nm beam
splitter, and a 590-nm long-pass emission filter.
Statistical Analysis
To rank the bulls accurately on their ability to bind to the zona pellucida
of the oocyte, an analysis of variance (ANOVA) model to evaluate variables in
the assay was used. The statistical model was as follows:
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Calculation of Breeding Data
For 15 bulls, lifetime 60- to 90-day nonreturn to estrus data were obtained
from a range of 1517 to 167855 services with an average of 84686 services per
bull. For 8 bulls, a competitive index was calculated from a heterospermic
insemination study (Saacke et al,
1980). For accessory spermatozoa, the mean number of accessory
spermatozoa per 6-day-old ovum or embryo nonsurgically recovered after
homospermic insemination to 1 of 3 bulls was obtained (n = 117, 39 ova or
embryos per bull; Dalton et al,
2001). The nonreturn rate, competitive index, and accessory
spermatozoa number were then used to assess the correlation of each with the
ranking of the bulls by in vitro zona-binding ability.
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Results |
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Using ANOVA to analyze the binding data for each bull, the estimable least squares means for the number of spermatozoa from each bull that were bound to oocytes was determined. The pairwise competitive assay was able to effectively rank the 15 bulls on their ability to bind to the zona pellucida (R2 = 0.84, Figure 1A). The 15 bulls used in this study had a relatively wide distribution (0.57-0.76) of nonreturn to estrus rates (Figure 1B). However, there was no significant correlation (r = -0.04, R2 = 0.002) between the ability of a bull's spermatozoa to bind to the zona pellucida and the fertility of that bull on the basis of nonreturn rates (Figure 1C).
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Relationship of Competitive Zona Binding to Fertility Calculated by
Competitive Insemination
Although competitive zona-binding ability was not related to nonreturn
rate, nonreturn rate only measures uncompensable traits because excessive
numbers of spermatozoa are inseminated. A second limitation is that
substandard semen samples are not available commercially, thereby narrowing
the range of semen quality available. In consideration of these facts, we
performed a second study using semen from 8 bulls that was collected for
previous experiments (Saacke et al,
1980). The fertility of these bulls was determined by competitive
insemination (Saacke et al,
1980). Semen from pairs of bulls was inseminated, and paternity of
offspring was determined by genetic markers or blood typing. A competitive
index was then calculated to determine relative fertility. Fertility
determined in this manner would include the impact of both compensable and
uncompensable seminal traits (Saacke et
al, 2000).
Estimable least squares means of the average number of spermatozoa bound to the zona pellucida was calculated. Using a series of pairwise comparisons, bulls were ranked by zona-binding ability effectively (R2 = 0.67, Figure 2A). These bulls also had a wide distribution of their heterospermic fertility competitive index (Figure 2B).
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The zona-binding ability of spermatozoa was not correlated to fertility, as assessed by calculating the competitive index in a heterospermic study (r = 0.29, R2 = 0.08, Figure 2C). Defects in zona binding were not frequent enough to allow the detection of a correlation between zona binding and fertility.
Relationship of Competitive Zona-Binding Ability to Accessory
Spermatozoa
Despite the lack of correlation between zona-binding ability and fertility
across a group of bulls, there may be occasional individual bulls with either
a competitive advantage or a disadvantage in zona binding that may affect
fertility. In this regard, one very interesting bull has been studied
(Dalton et al, 2001).
Significantly greater numbers of his spermatozoa are found as accessory
spermatozoa on embryos after insemination than on embryos of control
spermatozoa. We determined if more of his spermatozoa bound to oocytes in
vitro when using a competitive assay.
Samples from the test bull and the 2 control bulls were compared by the in vitro competitive zona-binding assay (Figure 3A). These 3 samples were ranked effectively by zona-binding ability in the competitive assay (R2 = 0.87) (Figure 3B). However, spermatozoa from the bull that had more accessory spermatozoa on embryos did not bind to the zona pellucida more frequently in vitro (Figure 3C).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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A competitive zona-binding assay was able to rank bulls efficiently on the basis of zona-binding ability. The ranking established from the competitive-binding assay was not correlated to either nonreturn to estrus rate or competitive index calculated from in vivo heterospermic inseminations.
The relationship between zona binding and fertility has been debated for some time. Two studies that tested whether zona binding was related to the 56-day nonreturn rate gave contrary answers (Fazeli et al, 1997; Zhang et al, 1998). One reason may be that in these studies, zona binding was calculated by 2 different methods. The Fazeli study used an index when recording zona binding, calculated by dividing the number of spermatozoa bound to oocytes for a test sample by the number of control spermatozoa bound to the oocytes, and did not detect any correlation between the index and nonreturn rate. In the Zhang study, both the absolute number of spermatozoa bound to oocytes and a binding index were recorded. Zhang et al (1998) found a correlation when calculating binding by absolute numbers but not when using an index. The studies described herein report zona-binding measurements as an average of each pairwise competition. Because we used a competitive assay, no correction for oocyte variability was needed. When using this method of estimating zona-binding ability, we did not detect a correlation between zona binding and 56-day nonreturn rates.
Other factors may account for the lack of correlation between zona binding measured by a competitive assay and nonreturn rate. One factor is that uncorrected non-return rates are influenced by many factors (ie, age of female and number of inseminations; Grossman et al, 1995). The influence of these factors has been corrected to yield the adjusted nonreturn rate values used in this study. Another factor to consider is that high dosages of spermatozoa are used for artificial insemination and that, in this situation, usually only bulls whose spermatozoa have uncompensable defects will have reduced fertility detectable by recording nonreturn rates. Considering this, it was important also to use a fertility measurement that could detect both uncompensable and compensable defects. In addition, analysis of a greater number of ejaculates may improve the relationship of nonreturn rate and zona binding.
In the second study, we assayed 8 bulls whose fertility was calculated from heterospermic inseminations, which detects uncompensable and compensable traits. Another advantage of measuring fertility by heterospermic insemination is that it is more reliable than nonreturn rates when using a limited number of inseminations (Robl and Dziuk, 1988). Fertility determined by this method was not correlated with in vitro binding. The nonsignificant correlation suggests that zona binding is not a step that frequently reduces bovine fertility. A later step in the fertilization pathway, such as zona penetration, membrane fusion, or oocyte activation, or an earlier step that does not influence zona binding may be defective.
Although we did not detect a significant correlation between zona binding and fertility, we also wanted to determine if there was a relationship between the number of accessory spermatozoa on the zona pellucida of embryos or ova and fertility. The number of accessory spermatozoa may be a reasonable indicator of spermatozoal transport, zona-binding ability in vivo, or both; however, spermatozoa from 1 bull that provided high numbers of accessory spermatozoa did not have a competitive advantage measurable by in vitro zona binding (Dalton et al, 2001). Spermatozoa from that bull may not have an advantage in zona binding, but instead, a higher proportion of his spermatozoa may be transported through the female reproductive tract to the site of fertilization. Transport through the female tract is an important variable in determining male fertility (Parrish and Foote, 1985). Indeed, in heterospermic insemination experiments, spermatozoa from 1 male can be predominant in compartments of the female reproductive tract several hours after insemination (Overstreet et al, 1978).
In humans, zona binding is often defective in infertile males (Liu and Baker, 2000). This competitive-binding assay may be useful in human fertility. However, it is difficult to determine if zona-binding ability is related to human fertility due to the difficulty of collecting accurate fertility data.
Although a competitive zona-binding assay was accurate in ranking bulls on binding ability, zona binding was not correlated with fertility measurements or zona binding in vivo. In order to estimate the fertility potential of semen samples, spermatozoal transport and steps postzona binding need to be studied. Carefully designed studies should allow the identification of specific steps that are the most common defects that reduce male fertility.
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Acknowledgments |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Braundmeier AG, Miller DJ. The search is on: finding accurate molecular markers of male fertility. J Dairy Sci.2001; 84:1915 -1925.[Abstract]
Collin S, Sirard M, Dufour M, Bailey JL. Sperm calcium levels and chlortetracycline fluorescence patterns are related to the in vivo fertility of cryopreserved bovine semen. J Androl.2000; 21:938 -943.[Abstract]
Dalton JC, Nadir S, Bame JH, Noftsinger M, Nebel RL, Saacke RG. Effect of time of insemination on number of accessory sperm, fertilization rate, and embryo quality in nonlactating dairy cattle. J Dairy Sci. 2001;84:2413 -2418.[Abstract]
Dziuk PJ. Factors that influence the proportion of offspring sired by a male following heterospermic insemination. Anim Reprod Sci. 1996;43:65 -88.
Fazeli AR, Zhang BR, Steenweg W, Larsson B, Bevers MM, van den Broek J, Rodriguez-Martinez H, Colenbrander B. Relationship between sperm-zona pellucida binding assays and the 56-day nonreturn rates of frozen-thawed bull semen. Theriogenology.1997; 48:853 -863.
Flowers WL. Management of boars for efficient semen production. J Reprod Fertil.1997; 52(suppl):67 -78.
Grossman M, Koops WJ, den Daas JH. Multiphasic analysis of reproductive efficiency of dairy bulls. J Dairy Sci.1995; 78:2871 -2876.[Abstract]
Ibrahim NM, Glibert GR, Loseth KJ, Crabo BG. Correlation between clusterin-positive spermatozoa determined by flow cytometry in bull semen and fertility. J Androl.2000; 21:887 -894.[Abstract]
Liu DY, Baker HW. Defective sperm—zona pellucida interaction:
a major cause of failure of fertilization in clinical in-vitro fertilization.
Hum Reprod.2000; 15:702
-708.
Marks JL, Ax RL. Relationship of nonreturn rates of dairy bulls to binding affinity of heparin to sperm. J Dairy Sci.1985; 68:2078 -2082.
Miller DJ, Demers JM, Braundmeier AG, Behrens ML. The use of 2
fluorescent dyes to identify sperm in a competitive binding assay to oocytes.
J Androl. 1998;19:650
-656.
Overstreet JW, Cooper W, Katz DF. Sperm transport in the reproductive tract of the female rabbit. II. The sustained phase of transport. Biol Reprod.1978; 19:115 -132.[Abstract]
Parrish JJ, Foote RH. Fertility differences among male rabbits determined by heterospermic insemination of fluorochrome-labeled spermatozoa. Biol Reprod.1985; 33:940 -949.[Abstract]
Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod.1988; 38:1171 -1180.[Abstract]
Robbins RK, Saacke RG, Chandler PT. Influence of freeze rate, thaw rate and glycerol level on acrosomal retention and survival of bovine spermatozoa frozen in French straws. J Anim Sci.1976; 42:145 -154.
Robl JM, Dziuk PJ. Comparison of heterospermic and homospermic inseminations as measures of male fertility. J Exp Zool. 1988;245:97 -101.[Medline]
Saacke RG, Dalton JC, Nadir S, Nebel RL, Bame JH. Relationship of seminal traits and insemination time to fertilization rate and embryo quality. Anim Reprod Sci.2000; 60-61:663 -677.
Saacke RG, Vinson WE, O'Conner M, et al. The relationship of semen quality and fertility: a heterospermic study. In: Proceedings of the Eighth Technical Conference on Artificial Insemination and Reproduction. Milwaukee, Wis. 1980:71 -78.
Shimizu Y, Kodama H, Fukuda J, Tanaka T. Evidence of proacrosin
molecule abnormality as a possible cause of low acrosin activity and
unexplained failure of fertilization in vitro. J
Androl. 1997;18:281
-288.
Zhang BR, Larsson B, Lundeheim N, Rodriguez-Martinez H. Sperm characteristics and zona pellucida binding in relation to field fertility of frozen-thawed semen from dairy AI bulls. Int J Androl.1998; 21:207 -216.[Medline]
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