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METHOD FOR MEASURING MOBILITY OF
SPERM
FIELD OF THE INVENTION
This invention concerns a method of improving breeding 5 and poultry operations by identifying highly fecund males within a population of males having previously unknown reproductive potential.
BACKGROUND OF THE INVENTION
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Commercial breeding and egg-laying operations have long sought to identify highly fecund males that would be effective breeders. A "fecund" male is one capable of producing many offspring, as opposed to a "fertile" male, which is merely capable of producing offspring. The iden- 15 tification of highly fecund animals is very important to the profitable operation of commercial breeding businesses. High producing males, whose sperm reliably fertilizes female eggs, improve the productivity and profitability of breeding and laying operations. A variety of techniques have 20 been used to improve male productivity, including artificial insemination and semen evaluation.
Aside from relying on historical data demonstrating fecundity of males, many techniques have been developed to attempt to predict fecundity of a male from examination of 25 a seminal sample. Tests evaluating percentages of motile sperm, ion characteristics of sperm, or membrane integrity have been moderately useful but not reliably predictive of effectiveness as a breeder. Morphological examination and characterization of sperm has also been helpful, because 30 morphologically normal sperm are more likely to be fertile. However tests evaluating these characteristics of sperm have been much more effective at distinguishing sub-fertile males from normal males, without identifying the relatively few males (top 30%) who will be the most effective breeders. 35 Given the many morphologic, anatomic, and physiologic variables that are involved in fertilization, some experts in this field have declared that it does not appear possible to predict fertility of a male from a semen sample in the absence of historical breeding data. 40
The identification or isolation of motile sperm has been extensively studied in both human and veterinary medicine. The widespread adoption of assisted insemination, such as intrauterine insemination, gamete intrafallopian transfer (GIFT), or in vitro fertilization (IVF) for infertile human 45 patients, has particularly stimulated the development of techniques for isolating, from an individual donor, a subpopulation of sperm that has increased fertilizing capacity. One such assay involves layering a liquid medium on top of a semen sample, and allowing the sperm to swim-up into the 50 medium. The sperm are then recovered and evaluated for concentration, motility and morphology. Almagor et al., /. AssistedRepro. and Gen. 10:261-265 (1993). Alternatively, sperm for in vitro fertilization can be selected by centrifuging sperm samples through gradients of Percoll solution. 55 Chan et al, Fertil. and Steril. 61:1097-1102 (1994).
The quality of sperm from oligospermic and asthenospermic males has also been improved by allowing sperm in a seminal specimen to migrate through a tube that contains polysaccharide beads that develop rough surface ridges after 60 hydration. Abnormal sperm are delayed from penetrating the column's pores by these ridges, which selectively allows motile sperm to be filtered out of the column. Ohashi et al., Fertil. and Steril. 57:866-870 (1992). The authors of this study reported that it was effective at slightly improving 65 conception in infertile couples, resulting in conception in 2 out of 21 couples where the male was oligospermic.
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Motile sub-populations of human sperm from an individual donor have also been isolated by suspending the sperm in a hypoosmotic medium (210 mOsm/kg) that is overlaid on an isolation medium of higher osmolality (360 mOsm/kg), that in turn overlays a recovery medium having an osmolality (290 mOsm/kg) identical to that of human uterine fluid. Motile sperm swim downward from the hypoosmotic medium toward the physiologically suitable medium (290 mOsm/kg), through interface barriers, to isolate motile sperm for IVT or GIFT. Lin et al., Arch. Androl. 27:177-184 (1991). This method is said to selectively isolate highly motile sperm from ejaculates, but it is not taught to be useful in the identification of highly fecund males.
The techniques described above are used to help improve fertilization capacity of an individual (usually oligospermic) male, instead of selecting breeding stock. Moreover, many of the findings from mammalian fertility research may not be applicable to avian breeding operations, such as poultry farms, because of substantial differences between mammalian and avian anatomy and physiology. In particular, mammalian sperm requires a biochemical activation step known as "capacitation" before fertilization can occur. Hence morphologically and functionally normal appearing mammalian sperm may be incapable of fertilization because of biochemical subtleties not measured by many morphologic and functional assays.
Fertilization in poultry also differs from mammals because spermatozoal sequestration in the oviduct's spermstorage tubules allows a hen to lay multiple fertilized eggs after a single insemination. When a hard-shelled egg is laid, the vaginal sphincter relaxes and allows the oviduct to become momentarily patent. If sperm are sequestered in the storage tubule when the oviduct opens, fertilization may occur if sperm are released concurrently with relaxation of the sphincter, and passively transported upwardly through the oviduct by muscle contraction. These differences between mammalian and avian reproduction make it difficult to extrapolate from the results of mammalian assays, and draw reliable conclusions about the use of mammalian assays in avian species such as chickens and turkeys. Moreover, these tests have been designed to assess fertility and not fecundity.
A variety of methods have previously been proposed for the objective measurement of sperm motility in animals. In one such assay, sperm cells from a test specimen are allowed to "swim-up" into a clear medium from a concentrated sperm suspension at the bottom of an optical cuvette. Highly motile sperm cause a time-dependent increase in turbidity of the medium, which can be used to determine a fraction of rapidly moving sperm and an average velocity of the sperm. The changing turbidity of the medium is recorded by a spectrophotometer as an increase in absorbance. Sokoloski et al., Fertil. and Steril. 28:1337-1341 (1977). This reference states that no firm correlation has been established between motility and fertilizing capacity.
A swim-up technique for evaluating ram semen motility is also described in Suttiyotin and Thwaites, /. Repro. Fertil. 97:339-345 (1993). In this assay, ram semen is layered at the bottom of disposable cuvettes, and overlaid with a variety of clear media. Turbidity of the overlying medium is measured using a calorimeter, and the turbidity of the medium is said to correlate with motility. This reference does not disclose a use for this motility data.
PCT publication WO 95/29983 (Hammerstedt et al.) discloses a method for testing the potential fertility of spermatozoa in a sample by incubating an aliquot of the
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sperm sample with a binding protein extracted from native vitelline membranes removed from chicken or turkey eggs. The number of sperm that bind to the protein are then determined by examining the surface of a substrate on which the protein is coated. The PCT publication notes that there 5 is a direct and linear correlation between sperm binding to the protein and the fertility of the spermatozoa in the sample. This assay is an in vitro test designed to simulate in vivo binding of the sperm to egg proteins, and the assay therefore provides an index of binding capacity of the sperm. This 10 isolated measure of fertilization capacity does not alone provide a highly predictive measure of fertilization capacity of a male from which the sample is obtained.
In spite of years of research into the identification of highly fecund avian males, and the motivation of the com- :5 mercial need for such identification, a reliable assay has not been found that predicts fecundity in males not having a breeding record. These failures have provoked pessimism among expert andrologists, who have predicted that such an assay will never be found for any species. Amman and 20 Hammerstedt, J. Androl. 14:397^106 (1993).
It is therefore an object of this invention to provide a quick and reliable assay for the ready identification of highly fecund avian males who do not already have a breeding record. 25
Another object of the invention is to provide a device that is capable of performing such a method in an economical and efficient manner.
These and other objects of the invention will be better 30 understood by reference to the following drawings and detailed description.
SUMMARY OF THE INVENTION
The foregoing objects are achieved by a method of 35 identifying highly fecund males by evaluating the mobility of populations of sperm from a male having unknown fecundity. The term "mobility" refers to movement of populations of sperm from one three dimensional medium to another, as contrasted to "motility" which refers merely to a 40 proportion of sperm that move within a field of view. Although motility is an indication of spermatozoal viability, it has been found that motility provides inadequate information about the fecundity of a male from which the semen sample is obtained. The present invention therefore instead 45 measures migrations of populations of sperm through a three-dimensional medium, or movement of sub-population of sperm from one three-dimensional compartment into another. The inventor has found this assay to be superior for identifying highly fecund males. 50
In the assay of the present invention, a semen sample containing avian sperm from a male test animal is diluted in an isotonic, buffered diluent to provide a test specimen. A liquid barrier medium, adjacent the test specimen, is sufficiently wide and dense or viscous that migration of sperm 55 from the test specimen is slowed when the sperm enters the dense medium. Highly mobile sperm have been found to migrate from the isotonic diluent into and through the dense barrier medium faster than less mobile populations of sperm from less fecund males. The dense medium is preferably 60 maintained at an optimum physiologic temperature for mobility of the sperm. Migration of sperm into and/or through the dense medium continues for a sufficient period of time to allow the assay to distinguish sperm from highly fecund males from sperm of less fecund males. The number 65 of sperm that migrate into the separation medium for highly fecund males will, for example, be at least one standard
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deviation above the mean number of sperm from males, within a base population of 100 or more males, that are less likely to be highly fecund.
In a more particular embodiment of the method, the barrier medium is a dense medium layered on the bottom of a container, and a less dense, immiscible test specimen is placed on top of the barrier medium. The test specimen medium includes semen from a male avian test subject and a diluent that is isotonic with the specimen of sperm and buffered to a physiologic pH. The test specimen and barrier medium are sufficiently immiscible to maintain their separation along the interface between them during the time period in which the assay is performed, and longer. The density of the barrier medium is sufficiently greater than a density of the test specimen to allow highly mobile sperm to migrate into the barrier medium more quickly than sperm that are not highly mobile. The barrier medium is maintained at a preselected temperature of about 40°-41° C. for a sufficient period of time to allow highly fecund males to be identified.
The method is particularly adapted for rapid identification of highly fecund males in a commercial breeding operation by automatically quantitating migration of sperm into or through the barrier medium. Automatic quantitation may be performed either by measuring increases in optical density of the barrier medium as sperm migrate into the barrier medium, or by collection of sperm on a collection member after the sperm migrate through the barrier medium. The collection member preferably includes an extract from a vitelline membrane of turkey or chicken eggs to which poultry sperm adhere. The barrier medium is preferably Accudenz [N,N'-bis(2,3-dihydroxypropyl)-5-N-2,3dihydroxypropylacetamido)-2,4,6-triido-isophthal amide], preferably 2-10% (wt/vol) in an aqueous solution, more preferably about 4—8% (wt/vol) of Accudenz per volume of solution.
The density of the barrier medium is preferably at least 1.005, for example 1.005 to 1.075. Particularly disclosed embodiments have calculated densities of 1.04 to 1.05. The density of the test specimen medium is less than the density of the barrier medium, and is more preferably about the density of water (for example 1.000 to 1.005) or less. In disclosed embodiments there is also a difference in density of about at least 0.005 between the specimen medium and the barrier medium, more particularly a difference of about 0.005 to 0.070, and most particularly a difference of at least 0.035.
The invention also includes a device for selecting highly fecund males, in which a container is provided with a temperature regulator for maintaining the container at a preselected physiologic temperature at which sperm mobility is optimal. A volume of liquid barrier medium is provided in the container, and a volume of liquid test specimen medium is layered on top of the barrier medium in the container. An automated device then quantitates the number of sperm that migrate into or through the barrier medium, to provide an index of the fecundity of the test animal. Specifically, the quantity of sperm detected by the automated device permits identification of highly fecund males.
In some embodiments, the automated device is a spectrophotometer that measures the optical density of the barrier medium. The optical density of the separation medium will increase linearly in direct proportion to the quantity of sperm that migrate into the medium. Alternatively, the automated device is a collection member below the barrier medium to which sperm adhere after they migrate through
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