WO1995004276A1

WO1995004276A1 - Physiological and isotonic buffer compatible with atp-luciferase bioluminescence reaction

Info

Publication number: WO1995004276A1
Application number: PCT/CA1994/000408
Authority: WO
Inventors: Abdullah K. Kirumira; Hermes K. W. Chan
Original assignee: Firezyme Diagnostic Technologies Ltd.
Priority date: 1993-07-28
Filing date: 1994-07-28
Publication date: 1995-02-09
Also published as: AU7343794A

Abstract

A novel formulation is provided for a buffer which uses sugars instead of salts, to provide an isotonic environment which is osmotically balanced with living cells but causes minimal inhibition of the ATP-luciferin-luciferase bioluminescence reaction. The buffer also nourishes and sustains the cells as shown by prolonged survival in the buffer without cell lysis. The buffer was developped for use in a sperm viability test based on intracellular ATP determination, but is suitable for use in viability assays including tissue cultured cells, homopoietic and other tissue derived cellular suspensions.

Description

PHYSIOLOGICAL AND ISOTONIC BUFFER COMPATIBLE

WITH ATP-LUCIFERASE BIOLUMINESCENCE REACTION

BACKGROUND OF THE INVENTION

The invention relates to a novel buffer formulation, more particularly to a buffer which uses sugars instead of salts to provide an isotonic environment which is osmotically balanced with living cells but causes minimal inhibition of the ATP-luciferin-luciferase

bioluminescence reaction.

Living cells in vitro require a medium which is osmotically balanced with the cytoplasm to prevent leakage of metabolites and damage to the cells. Typically such physiological media are salt solutions, such as Hank's PBS (Phosphate-Buffered Saline) or Ringer's solution. However, high ionic strengths of these solutions affect the luminescent output of the firefly luciferase and luciferin

(Denburg et al, Arch, of Biochem. Biophys., vol. 141, p. 668-675, 1970). In an ideal luminescence assay, the endpoint sought is the intracellular ATP of a cell

suspension such as blood cells, sperm, or bacteria; this would be determined by subtracting the extracellular ATP, measured in buffer alone, from total ATP, including the ATP extracted from the suspended cells. If the buffer used for the blank is not osmotically balanced with the cells, ATP will leak and increase the blank reading. If the buffer inhibits the luciferase and luciferin, light output will be reduced. As recently as 1987, Gottlieb et al (Fertility & Sterility, vol. 47, No. 6, p. 992) were unable to detect significant amounts of ATP in blank seminal specimens, due to lysis of spermatozoa by the EDTA buffer added. The use of EDTA in the TRIS buffer reported by Gottlieb et al inhibits the ATP-degrading phosphatases in seminal fluid and stabilizes the ATP concentration by protecting it from degradation by those enzymes.

SUMMARY OF THE INVENTION

When a sample, such as seminal spermatozoa, where intracellular ATP is to be measured contains free soluble exogenous ATP, this ATP has to be eliminated or determined to ascertain an accurate measurement of intracellular ATP. Moreover, the quantity of extracellular ATP itself or changes in this quantity may provide additional information about the sample. Typically, in the determination of intracellular ATP, the extracellular ATP is eliminated first.

A commonly used technique is the addition of an ATP-ase enzyme, such as apyrase, to break down the free ATP outside the cells. The intracellular ATP is then extracted and determined following inactivation of the residual ATP hydrolyzing enzyme. A major drawback of this technique is the additional processing time required for adding and removing the ATP-ase enzyme, making it tedious and

time-consuming for routine use in the laboratory.

Yet another approach described is removing of cells from the liquid media by centrifugation, followed by washing and resuspending cells in fresh media prior to ATP extraction. This approach may cause adverse changes in intracellular ATP level and in the overall Adenylate Energy Charge of the cell. Furthermore, centrifugation allows release of ATP-ase enzymes that destroy any exogenous ATP, making it impossible to ascertain the free ATP originally present in the sample.

Filtration is another approach that has been attempted for separating cells. This also suffers from the same effect of subjecting cells to a stress that causes a sudden drop in ATP contents and Adenylate Energy Charge as well as being more cumbersome.

In a recent study, Gottlieb et al (ibid) attempted to determine free ATP in seminal plasma by addition of TRIS-EDTA buffer, devoid of an ATP extracting agent. Measurable amounts of ATP were reported concomitant with impaired sperm motility. It was concluded that release of some ATP from spermatozoa was induced by the EDTA buffer used.

Accordingly, it is an object of the preesent invention to provide an isotonic buffer which sustains the structural and physiological integrity of homopoietic. somatic and reproductive cells, thereby preventing leakage of intracellular analytes and which also causes minimal inhibition of ATP-luciferin-luciferase reaction.

Another object of the present invention is to provide an improved method for determining and correcting exogenous (intercellular) ATP in biological specimens, which substantially prevents or alleviates one or more of the disadvantages of methods known in the prior art.

A further object is to provide a method for quantifying number of viable cells in biological specimens by utilizing a bioluminescent reaction to measure

intracellular ATP when the sample analysed also contains free exogenous ATP.

The present invention allows a more accurate determination of intracellular ATP in living cells by means of an isotonic physiological buffer which allows an

exclusive determination of exogenous ATP. This value is then subtracted from the total ATP measured following complete lysis of the cells by an appropriate extractant and addition of the buffer medium in a parallel but

otherwise identical assay.

Thus, according to one aspect, the invention provides an isotonic buffer sustaining the structural and physiological integrity of homopoietic, somatic and reproductive cells, said buffer having a pH of from about 7.0 to about 8.0 and comprising N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid], ethylenediaminetetraacetic acid, and a sugar.

According to another aspect, the invention provides a method of measuring intercellular ATP in

somatic, homopoietic or reproductive cells, which method comprises: suspending cells in a buffer maintaining the structural and physiological integrity thereof, reacting a sample of the suspended cells with a luciferin-luciferase reagent, measuring a bioluminescent signal generated by the sample, and calculating the amount of the intercellular ATP in the sample from the bioluminescent signal. According to yet another aspect, the invention provides a method of measuring changes in intercellular ATP in sematic, homopoietic or reproductive cells, which method comprises: suspending cells in an isotonic buffer

maintaining the structural and physiological integrity thereof, reacting a first sample of the suspended cells with a luciferin-luciferase reagent, measuring a first bioluminescent signal generated by the first sample, after a predetermined period of time from reacting the first sample, reacting a second sample of the suspended cells with the luciferin-luciferase reagent, measuring a second bioluminescent signal generated by the second sample, and calculating the change in the amount of intercellular ATP from the difference of the first and the second

bioluminescent signals.

According to still another aspect, the invention provides a method of measuring intracellular ATP in or determining viability of somatic, homopoietic or

reproductive cells, which method comprises: suspending cells in a buffer maintaining the structural and

physiological integrity thereof, reacting a first sample of the suspended cells with a luciferin-luciferase reagent, measuring a first bioluminescent signal generated by the first sample, treating a second sample of the cells with an ATP extracting agent, suspending the second sample in the buffer, reacting the second sample with a luciferin-luciferase reagent, measuring a second bioluminescent signal generated by the second sample, and calculating the amount of the intracellular ATP in the samples from the first and the second bioluminescent signals, and, if required, determining the viability of cells from the calculated amount of the intracellular ATP.

The buffer according to the invention provides an isotonic environment which is osmotically balanced with the cytoplasm of living cells. Such osmotic balance is usually achieved in solutions containing inorganic salts. However, high ionic strength of these solutions adversely affects the luminescent output of ATP-luciferin-luciferase reaction. The reduced light output makes impossiblea reliable

determination of intercellular (extracellular) ATP present in cell samples using a bioluminescent reaction. It has now been found that a satisfactory osmotic balance may be achieved in buffer solutions substantially free of inorganic salts, by replacing the latter with an appropriate amountof sugars. As an additional advantage, sugars present in the buffer nourish and sustain the cells, prolonging their survival without cell lysis. More importantly, such buffers cause minimal inhibition of the ATP-luciferin-luciferase

bioluminescent reaction, thus making possible the

determination of intercellular ATP. The amount of

intercellular ATP, when substracted from the similarly determined amountof total ATP, including the ATP extracted from the cells, provides an improved measure of the

intracellular ATP and, as a result, an improved measure of viability of the cells. The concentration of intercellular ATP in cell samples is a balance of the rate of its production, primarily by damaged cells, and its consumption by ATPase enzymes, which come from damaged and lysed cells, as well as membrane-bound ATPases or normal cells, as well as other consumption. Thus the proportion of total ATP which is intercellular is a measure of the proportion of damaged cells in the sample; the change of intercellular ATP immediately following dilution is a measure of the processes of production and consumption.

The buffer of invention is based on an organic acid and a salt thereof and, for compatibility with living cells, has a pH of from about 7 to about 8, preferably about 7.75. N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] (HEPES) is a preferred acid componentof the buffer, in a concentration of from about 25 mmol/L to about 100 mol/L, preferably about 25 mol/L. The salt of the acidis preferably sodium salt.

The sugar component of the buffer is preferably a

D-hexose, most preferably D-glucose. The latter should be present in a concentration of from about 2% bw to about 20% bw, preferably from about 2% bw to about 8% bw, more preferably from about 4% bw to about 6% bw, most preferably about 5% bw.

The buffer of the invention also includes a phosphatase inhibitor, which component inhibits ATD-degrading phosphatases

present in an original sample of cells or liberated by treating the sample with an ATP extracting agent.

Ethylenediaminetetraacetic acid (EDTA) is preferably used for this purpose in concentration from about 1 mmol/L to about 3 mmol/L, preferably about 2 mmol/L.

Even though the buffer of the invention was developed for use in a sperm viability test based on intracellular ATP determination, it is suitable for use in viability assays including tissue cultured cells,

homopoietic and other tissue derived cellular suspensions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows ATP calibration curves in various buffers without added TCA extractant,

Figure 2 shows ATP calibration curves in various buffers with added TCA extractant.

Figure 3 shows intracellular ATP bioluminescence as a function of proportion of viable semen.

Figure 4 shows correlation between viable sperm counts calculated by a reference method and a viable sperm count calculated by measuring intracellular ATP biolumin-escence.

Figure 5 shows stability of spermatozoa intracellular ATP following extraction in the FIB buffer, Figure 6 shows a time course of spermatozoa intracellular ATP bioluminescence monitored in the FIB buffer.

Fogire 7 shows mean ATP content of Blank sample for five bulls demonstrating increased blank vlaues as a result of shipping damages.

Figure 8 shows an example of published field trial data fitted with a hyperbolic fertility function. DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1

Preparation of the HEPES-EDTA Buffer

5.96 g of N-[2-Hydroxyethyl]piperazine-N'-2-ethanesulfonic acid] (HEPES), and 0.1 g of sodium azide and 0.832 g of ethylenediaminetetraacetate disodium salt (EDTA) were dissolved in double deinized (DDI) water and pH adjusted to 7.75 with 1N NaOH. The solution was then made up to a final volume of 1 litre with DDI water to provide a final concentration of 2mM EDTA in 25mM HEPES designated as the HEPES-EDTA buffer.

Preparation of the FIB Buffer

The FireZyme Isotonic Buffer was prepared by dissolving 55 g of analytical grade dextrose in 1 litre of the HEPES-EDTA buffer to provide a final concentration of dextrose at 5.5% (w/v).

Preparation of Hank's Balanced Salt

One unit the powdered medium supplied by Sigma Chemicals Co., Ltd. [H8389-1L] in one litre of DDI water containing 2mM EDTA and 0.1% (w/v) sodium azide. pH was adjusted to 7.75 using 1N NaOH.

Preparation of Physiological Saline

9.0 g of analytical grade NaCl were dissolved in

1 litre of DDI water containing 2mM EDTA and 0.1% (w/v) sodium azide. pH was adjusted to 7.75 using 1N NaOH.

Preparation of Phosphate Buffer [0.1 M]

17.82 g of Na₂HPO₄·2H₂O were dissolved in 1 litre of DDI water containing 2mM EDTA and 0.1% (w/v) sodium azide and 13.65 g of KH₂PO₄·2H₂O were dissolved in 1 litre of DDI water containing 2mM EDTA and 0.1% (w/v) sodium azide. Then 970 millilitres of the Na₂HPO₄·2H₂O solution was mixed with 30 millilitres of the KH₂PO₄·2H₂O solution to provide 1 litre of a 0.1 M phosphate buffer pH 7.75. Preparation of Tris Buffer p25mM]

3.94 g of Tris-HCl were dissolved in 1 litre of

DDI water containing 2mM EDTA and 0.1% (w/v) sodium azide. pH was adjusted to 7.75 using 1N NaOH.

Preparation of Glycine Buffer [0.1M]

111.52 g of Glycine-HCl were dissolved in 1 litre of DDI water containing 2mM EDTA and 0.1% (w/v) sodium azide. pH was adjusted to 7.75 using 1N NaOH.

Preparation of Tricine Buffer [25mM]

4.48g of Tricine were dissolved in 1 litre of DDI water containing 2mM EDTA and 0.1% (w/v) sodium azide. pH was adjusted to 7.75 using 1N NaOH.

Preparation of MOPS Buffer [25mM]

5.23 g of 3-[N-Morpholino]propanesulfonic acid)

(MOPS) were dissolved in 1 litre of DDI water. pH was adjusted to 7.75 using 1N NaOH.

Preparation of Citrate Buffer [0.15M]

38 g of Trisodium citrate were dissolved in 1 litre of DDI water containing 2mM EDTA and 0.1% (w/v) sodium azide. pH was adjusted to 7.75 using 1N NaOH.

EXAMPLE 2

Testing of Light Inhibition and Toxicity

of Various Buffer Compositions

The light inhibition was tested by adding pure

ATP to the buffer to be tested and comparing the reading with that of the control buffer (25 mM HEPES-EDTA buffer).

The buffers were then used to test sperm ATP, both total

ATP (RI) and extracellular ATP (Blank). The results of these tests are shown in Table 1. EXAMPLE 3

Testing of Buffer Effect on ATP Analysis by FireFlv

Luciferase

1. A luciferin-luciferase mixture was prepared from a freeze-dried preparation obtained from Sigma Chemical Co. Ltd., Saint Louis MO. The vial containing approximately 5 mg of the FireFly lantern extract was reconstituted in 5 mL of a diluent buffer (25mM HEPES pH 7.75).

2. 10 buffers commonly used in bioluminescence reactions and in maintenance of living cells, prepared as described in Example 1 (including the FireZyme

Isotonic Buffer (FIB)) were assembled and allowed to equilibrate to ambient temperature (Table 2).

3. The inhibitory effect of each buffer on the ATP

Luciferin-Luciferase reaction was then established by following a standardized protocol which involved: a. Taking 50μL from each of the 3 ATP standards

studied into 3 separate vials.

b. Adding 50μL of the test buffer containing the nucleotide extractant [10% Trichloroacetic acid (TCA)].

c. Adding 4 mL of the test buffer to the mixture as a neutralization and stabilizing diluent.

d. Then recording the bioluminescence signal

generated by reacting 100μL of the final mixture with 100μL of the Firefly Luciferin-Luciferase reagent in a luminometer. e. Percent inhibition was calculated using the bioluminescence signal in the 25 mM HEPES-EDTA medium as the reference control.

Table 2 illustrates results obtained in the presence of FIB buffer, in determining bioluminescence generated from a fixed concentration of ATP as compared to other commonly employed buffers. Inhibition of

ATP-Luciferin-Luciferase bioluminescence by the FIB buffer was minimal and comparable to the frequently used buffer media, for this reaction [i.e. Tricine, Tris, MOPS and

HEPES]. Concomitantly the compatibility of the FIB buffer with this bioluminescence reaction was found to be far much superior in comparison with Hank's Balanced Salt buffer and physiological Saline media frequently used for maintaining the structural and physiological integrity of living cells during analysis.

EXAMPLE 4

Determination of Intracellular ATP in Bovine Spermatozoa

To demonstrate the efficacy of using the FIB buffer to measure intracellular ATP in living cells, a duplicate set of freshly thawed bovine spermatozoa was analysed within the disclosed buffer medium with and without addition of a cell lysing agent. The ATP present in both samples was then monitored using the Firefly

Luciferin-Luciferase bioluminescence reaction, as a function of incubation time. An identical parallel set of experiments were then conducted using other commonly used buffers for comparison (Table 3). Procedure A

To establish the exogenous ATP level in the sample:

1. 50μl of freshly trawed bovine spermatozoa were

introduced into a polyethylene mixing vial.

2. 50μl of the test buffer, devoid of a lysing agent were then added to the sample and mixed.

3. 4ml of the FIB buffer were then added as a diluent and mixed.

4. The exogenous ATP level was then monitored by reacting 100μl of the final mixture with 100μl of the Firefly luciferin-luciferase reagent.

5. The bioluminescence signal generated was read in a luminometer and used to calculate total ATP, utilizing a standard curve constructed with known standards of ATP, analyzed under a similar protocol for each of the buffers tested (Figure 1).

Procedure B

To establish the intracellular ATP level in the sample a similar standard protocol was followed that involved:

1. Taking 50μl of a duplicate sample of freshly thawed bovine spermatozoa and introducing into a fresh mixing vial.

2. Adding 50μl of the test buffer containing the

nucleotide extracting agent [10% Trichloroacetic acid (TCA) and 2mM EDTA] followed by mixing.

3. Adding 4ml of the test buffer to the mixture as a

neutralization buffer. 4. The total ATP (exogenous + intracellular ATP) was then monitored by reacting 100μl of the final mixture with 100μl of the Firefly Luciferin-Luciferase reagent.

5. The bioluminescence signal generated was read in a luminometer and used to calculate total ATP, utilizing a standard curve constructed with known standards of ATP, analyzed under a similar protocol for each of the buffers tested (Figure 2).

Intracellular ATP was deduced by subtracting the exogenous ATP calculated in procedure A from the total ATP calculated in procedure B.

Table 3 illustrates results obtained using the FIB buffer in determining spermatozoa intracellular ATP in comparison with other commonly employed buffers. The FIB buffer shows a markedly higher ratio of [intracellular

ATP/exogenous ATP ratio] which accurately reflects what one would expect in a spermatozoa sample. The FIB buffer thus appears to allow a more accurate measurement of exogenous ATP by eliminating interference through leakage of

intra-cellular ATP into the extra-cellular environment. The ratios for other buffers in comparison were markedly lower suggesting that they allowed a significant leakage of ATP during the blank measurement procedure devoid of an the extractant. EXAMPLE 5

Quantitation of Viable Spermatozoa Utilizing Intracellular

ATP

Bioluminescence Using the FIB Buffer

To demonstrate the efficacy of using the FIB buffer for quantifying number of viable spermatozoa in specimens containing exogenous ATP, simulated mixtures were prepared by mixing a known amount fresh viable semen with increasing fractions of semen in which all spermatozoa had been rendered "dead" by several rapid freeze/thaw cycles. This gave a range of samples designated as 0%, 20%, 40%, 60%, 80% and 100%, where the percent refers to the

proportion of freshly extended viable semen in the sample formulation. Total ATP [ATP_T] in each sample was then determined by taking 50μL aliquots from each sample and treated with an equal volume of the TCA based extractant as previously described, prepared in the 25 mM HEPES-EDTA buffer. This allowed release of all spermatozoa

intracellular ATP. The mixture was then diluted with 4mL of the FIB buffer as a neutralization diluent. The total ATP [ATP_T] in the extract was then determined from the luminescence generated by mixing 100μL of the extraction mixture with 100μL of the FireFly Luciferin-Luciferase in a vial placed inside a luminometer measuring chamber.

In duplicate samples, exogenous ATP was determined as previously described in (Procedure B) in an identical protocol but devoid of the TCA nucleotide

extractant. The bioluminescence signal generated was used to calculated exogenous ATP [ATP_EX]. The difference between [ATP_T] and [ATP_EX] represented the spermatozoa intracellular ATP [ATP_IN] whose value was used to compute total viable sperm count in each sample, obtained by dividing [ATP_IN] with an established average ATP content per spermatozoa [ATPs].

The viable sperm count calculated from intracellular ATP bioluminescence measured using the unique characteristics of the FIB buffer, were compared with the viable count of a reference method utilizing a combination of a haemocytometer for total count and a differential DNA fluorescence staining method that determines the proportion of viable cells. The two procedures used for reference are well known and therefore not described herein. [Makler, A (1978); Fertil. Steril. Vol 30 pp. 313-318 and

Bilgili, S.F. and Renden, F.A. (1984); Poultry Science, Vol 63: pp. 2275-2277].

Figure 3 shows the relationship between fresh sperm concentration and bioluminescence generated from corrected intracellular ATP determined using the FIB buffer. The light intensity shows a highly significant positive relationship with viable sperm concentration

[R=0.989].

Table 4a and 4b summarize the results of intracellular ATP bioluminescence determinations made on semen mixtures with varying proportions of fresh and killed sperm. The total viable counts calculated from the determined intracellular ATP agrees very closely with the reference method. The coefficients of variation among successive determinations for each concentration of [fresh killed] semen ranged from 3-5% demonstrating one of the best reproducibilities reported for sperm analysis.

Total viable counts were measured in over 200 semen samples over a two months period. A good positive correlation between counts by the FireZyme SVT method and counts by the reference method was found. The coefficient of correlation was 0.906, with the equation of the

regression line being [y=2.199E-6 + 0.930x] (Figure 4). EXAMPLE 6

Stability of extracted ATP in the FIB buffer

A freshly thawed sample of bovine spermatozoa was extracted in FIB buffer as previously described (Procedure B). ATP bioluminescence from the sample was then measured in a series of 100μL aliquots taken from the extract at intervals of 30 min over a 2 hour period.

Figure 5 shows results of a study on the stability of ATP extracted from bovine semen using the FireZyme FIB buffer. The ATP concentration remained constant over a period of time. The results show the ATP content after 2 hours, was on the average 102% of initial values (range 98% to 105%; n=7, median 102%).

The signal stability of extracted ATP

demonstrated by using the FIB buffer is significant in that, it can allow batched sample processing with no critical time steps. The measurement process therefore becomes tolerant of the interruptions normal in a busy production or laboratory environment. We have observed in this laboratory that when extraction mixtures were stored at -20C, there was no notable change in ATP concentration for over 5 weeks. This provides a busy laboratory further flexibility in sampling, processing and storing or

transporting a large number of specimens for later

analysis.

EXAMPLE 7

Kinetics of ATP bioluminescence in the FIB buffer

For a more accurate determination of ATP, the bioluminescence reaction must give a constant light emission during the experiment. Figure. 6 shows the time course of light emission following extraction and

continuous monitoring of the light signal in the FIB buffer. The results illustrate a relatively constant rate of light emission with time. This demonstrates a minimal consumption of ATP during the assay. The peak light output declined by less than 4% per minute. This can be

attributed to the protection of the measured ATP from degradation conferred by the FireZyme FIB buffer. The new buffer therefore provides an improvement over some of the methods previously reported where the peak light output is typically followed by a rapid decrease in light emission. This feature is advantageous in that it makes the

determination more tolerant of operator to operator variations in loading and initiation of the bioluminescence reaction.

EXAMPLE 8

Change in intercellular ATP as a measure of subtle damage Since intercellular ATP may be produced primarily from subtle membrane damage and leakage from otherwise viable cells, the intercellular ATP content provides a proportionate measure of subtle damage.

Five ejaculates from different bulls were prepared using standard methods in 0.25 ml straws and distributed to three laboratories, one being the laboratory of origin of these samples. Twenty-five straws from each ejaculate were tested in total; interassay variation ranged from 2% to 5% for the three labs. For comparison, the correlation for Total ATP between the three

laboratories was r = 0.997. The correlations for

intercellular ATP were r = 0.75 and 0.94 for the third laboratory (Acadia) with the laboratory of origin (UBI) and the second laboratory (U. of Guelph). Figure 7 shows these results plotted with UBI laboratory onthe X axis and the other laboratories on the Y axis; the error bars show the 95% confidence limits for the mean values plotted. The proportionate increase in Blank ATP content is different for each bull, and this increase is mirrored in both remote centers. There is no evidence that this increase is in experimental artifact, andit is far greater than the between-straws error for ATP determination using the FIB buffer of 0.075 mcg/ml.

The transport of frozen semen involves transferring the straws between liquid nitrogen containers, and exposure to room air has been shown to be deleterious to semen fertility, but it has taken many repeated exposures to establish this effect when normal measures of sperm quality are used (John Sullivan, American Breeders Service, personal communication). In this case, the semen was transported from UBI to U. of Guelph with only two transfers within a brief period of time. The semen tested at Acadia had been shipped through routine channels, involving multiple transfers ove a month. Thus the increase in intercellular ATP measured is a sensitive measure of subtle damage to the semen during handling.

EXAMPLE 9

Improved prediction of fertility of bovine semen using the FIB buffer

An ideal sperm viability test should predict the fertility of a semen sample better than the total

concentration of spermatozoa alone. The following example shows how use of the FIB Buffer improves fertility

prediction significantly by measuring both quality and quantity of viable semen.

The fertility of bovine semen is measured by the Non-Return Rate (NRR), the percentage of cows not being reinseminated after a fixed period, usually 59 days. For a given bull, the NRR is an asymptotic function of sperm number inseminated. The two attributes of the function are the asymptotic fertility achievable by that bull at infinite sperm numbers, alpha, and a parameter adjusting sperm number beta. Figure 8 shows how field trial data from several bulls fit onto one such a fertility function with normalized alpha and beta. Twenty frozen-thawed samples of semen from different bulls were tested using the method of Example 5; all samples were tested twice, and all diluted samples had two successive runs with luciferase and luciferin reagent to generate bioluminescence signal. These twenty samples had associated NRR based on extensive field trials. Three samples were apparently anomalous, and were initially excludedas outliers. The NRR values were fitted to the hyperbolic form of the fertility function using minimizatin of Chi-Square as the fitting metric. For this many degrees of freedom, a reduction of 3.07 unitsin Chi-square is needed to give a significantly better fit (P < 0.05). The initial results were as follows:

Parameter Alph Beta Chi² r

SVT Viable Sperm .688 5.94 77.13 .48

Sperm concentration .711 1.32 75.18 .50

Total motile sperm .708 2.32 74.69 .50

Cone, x Sqrt (motile) .713 1.99 73.80 .51

SVT Total ATP .704 2.90 70.41 .55 P<.01 SVT Blank ATP .719 24.8 61.78 .62 P<.001

Clearly Blank ATP is the best predictor of fertility of the sample, whereas Viable sperm. Total - Blank, is the worst. This paradoxical result was investigated further.

An additional variable was employed, Delta Blank, the change in the ATP reading of the Blank between two successive runs

immediately following dilution. When the FIB Buffer is added in this protocol, the ATP in the semen sample is diluted forty-fold, and the concentration then readjusts quickly to a new equilibrium value. Spermatozoa living in the FIB Buffer continue to leak ATP, and some of the

ATPases are not inhibited by the EDTA. As shown in Example 6, this new equilibrium persists for hours.

Due to the low value of Delta Blank, in uits of tenths of micrograms ATP per millilitre, it has a high Coefficient of Variation, estimated in this experiment as 30%. In the large interlaboratory experiment discussed in Example 9, Delta Blank was also found, but due to the large number of replicates it could be determined more precisely. The effect of runs is statistically valid, as it exceeds the error variance between runs for Total ATP, was

consistent on a bull-by-bull basis in all three labs and on all days.

When the fertility data for the 20 ejaculates discussed was fitted including Delta Blank as a variable, the best result was obtained with an equation of the form:

NRR = alpha* (1 + k B)*beta v/ (1 + beta v) In this case, the term including Delta Blank modifies the asymptotic fertility alpha, rather than the number of viable sperm v; thus it is measuring a quality of the ejaculate as a whole orthogonal to the numbers of viable spermatozoa. This quality may be related to the quality discussed by Pace et al. (J. Anim. Sci. 53(3),

693-701, 1981), which is affected by handling and reduces asymptotic fertility. For these samples and using v - SVT Total ATP, Chi² = 55.18, r = 0.675, a highly significant improvement in the prediction of fertility over the original form is achieved. This fit includes one of the outliers inthe data set fitted.

Thus the FIB buffer enables a better prediction of the fertility of frozen-thawed bull semen than conventional measures such as motility assessment and concentration.

The intercellular ATP and its change immediately following dilution are sensitive indicators of subtle damage to bull spermatozoa with a dramatic effect on fertility. This attribute of the FIB Buffer will make it useful as a research tool in improving sperm handling methods, as well as a prospective test of field fertility in the artificial insemination industry.

Claims

CLAIMS :

1. An isotonic buffer sustaining the structural and physiological integrity of homopoietic, somatic and

reproductive cells, said buffer being substantially free of inorganic salts and comprising an organic acid, a salt thereof, a phosphatase inhibitor and a sugar.

2. A buffer according to claim 1, wherein the organic acid is N-[2-hydroxyethyl ] piperazine-N' -[2-ethanesulfonic acid].

3. A buffer according to claim 2, comprising from about 25 mmol/L to about 100 mmol/L of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid].

4. A buffer according to claim 1, wherein the phosphatase inhibitor is ethylenediaminetetraacetic acid.

5. A buffer according to claim 4, comprising from about 1 mmol/L to about 3 mmol/L of ethylenediaminetetraacetic acid.

6. A buffer according to claim 1, having a pH of from about 7 to about 8.

7. A buffer according to claim 1, wherein the sugar is a D-hexose.

8. A buffer according to claim 7, wherein the D-hexose is D-glucose.

9. A buffer according to claim 8 comprising from about 2% by weight to about 10% by weight of glucose.

10. A buffer according to claim 9 comprising from about 2% by weight to about 8% by weight of glucose.

11. A buffer according to claim 10 comprising from about 4% by weight to about 6% by weight of glucose.

12. An isotonic buffer sustaining the structural and physiological integrity of homopoietic, somatic and reproductive cells, said buffer being substantially free of inorganic salts, having a pH of about 7.75 and comprising about 25 mmol/L of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid], about 2 mmol/L of

ethylenediaminetetraacetic acid and about 5% by weight of glucose.

13. A method of measuring intercellular ATP in somatic, homopoietic or reproductive cells, which method comprises:

- suspending cells in an isotonic buffer maintaining the structural and physiological integrity thereof,

- reacting a sample of the suspended cells with a

luciferin-luciferase reagent,

- measuring a bioluminescent signal generated by the sample, and

- calculating the amount of the intercellular ATP in the sample from the bioluminescent signal.

14. A method according to claim 13, wherein the isotonic buffer maintaining the structural and physiological

integrity of cells is substantially free of inorganic salts and comprises an organic acid, a salt thereof, a

phosphatase inhibitor and a sugar.

15. A method according to claim 13, wherein the isotonic buffer maintaining the structural and physiological

integrity of cells is substantially free of inorganic salts, has a pH of about 7.75 and comprises about 25 mmol/L of N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid], about 2 mmol/L of ethylenediaminetetraacetic acid and about 5% by weight of glucose.

16. A method according to claim 13, wherein the luciferin-luciferase reagent is a firefly luciferin-luciferase reagent.

17. A method of measuring intracellular ATP in or

determining viability of somatic, homopoietic or

reproductive cells which method comprises:

- suspending a first sample of cells in an isotonic buffer maintaining the structural and physiological integrity thereof,

- reacting the first sample of the suspended cells with a luciferin-luciferase reagent,

- measuring a first bioluminescent signal generated by the first sample,

- treating a second sample of the cells with an ATP extracting agent,

- suspending the second sample in the buffer,

- reacting the second sample with the luciferin-luciferase reagent,

- measuring a second bioluminescent signal generated by the second sample,

- calculating the amount of the intracellular ATP in the samples from the first and the second bioluminescent signals, and, if required,

- determining the viability of cells from the calculated amount of the intracellular ATP.

18. A method according to claim 17, wherein the isotonic buffer maintaining the structural and physiological integrity of cells is substantially free of inorganic salts and comprises an organic acid, a salt thereof, a

phosphatase inhibitor and a sugar.

19. A method according to claim 17, wherein the isotonic buffer maintaining the structural and physiological integrity of cells is substantially free of inorganic salts, has a pH of about 7.75 and comprises about 25 mmol/L of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid], about 2 mmol/L of ethylenediaminetetraacetic acid and about 5% by weight of glucose.

20. A method according to claim 17, wherein the luciferin-luciferase reagent is a firefly luciferin-luciferase reagent.

21. A method of measuring changes in intercellular ATP in somatic, homopoietic or reproductive cells following dilution, which method comprises:

- reacting a first sample of the suspended cells with a luciferin-luciferase reagent,

- measuring a first bioluminescent signal generated by the first sample,

- after a predetermined period of time from reacting the first sample, reacting a second sample of the suspended cells with the luciferin-luciferase reagent,

- measuring a second bioluminescent signal generated by the second sample, and

- calculating the change in the amount of intercellular ATP from the difference of the first and the second

bioluminescent signals.

22. A method according to claim 21, wherein the isotonic buffer maintaining the structural and physiological

phosphatase inhibitor and a sugar.

23. A method according to claim 21, wherein the isotonic buffer maintaining the structural and physiological

integrity of cells is substantially free of inorganic salts, has a pH of about 7.75 and comprises about 25 mmol/L of N-[hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]. about 2 mmol/L of ethylenediaminetetraacetic acid and about 5% by weight of glucose.

24. A method according to claim 21, wherein the luciferin-luciferase reagent is a firefly luciferin-luciferase reagent.