Field of the Invention

[0001 ] The present invention relates to a method for the separation/fractionation of X- and Y-chromosome-bearing spermatozoa and to the use of such spermatozoa in the insemination of female mammals. The present invention is preferably directed to cattle.

Background to the Invention

[0002] Sex pre-selection can be achieved either by sexing embryos or by the sexing of X- or Y-chromosome-bearing spermatozoa. Although good success rates in embryo sexing have been reported in livestock, sexing of spermatozoa is the preferred method as this avoids manipulation and wastage of embryos and can be integrated into the standard management of most dairy and beef enterprises. In addition, in embryo transfer programs, the use of sexed semen prevents embryonic wastage due to the production of embryos of the desired sex only.

[0003] The karyotype of cattle consists of 60 chromosomes, which include 29 pairs of autosomes and 1 pair of sex chromosomes. Sex chromosomes were first observed in mammals during the early nineteenth century.

[0004] Methods that have attempted to fractionate spermatozoa in the past have included head length measurements, electrophoresis of semen, density centrifugation, separation potential of pH and flow cytometry. However, no single method could be outlined as the most suitable given that every method has its own advantages and disadvantages. For example, while flow cytometry separates cells based on DNA content with an accuracy of about 90%, this technique requires very high equipment costs and sperm damage during the sexing and altered mRNA expression of embryos has also been reported in the peer reviewed literature using this method. On the other hand, the accuracy of density gradient methods and methods utilising separation potential of pH both separate X- and Y-chromosome-bearing spermatozoa, is lower than flow cytometry. Nonetheless, these methodologies appear to be cost-effective and result in less damage to the sperm, indicating their better fertilization potential.

[0005] The demand for semen sexed using flow cytometry would be far greater if the fertility of the semen was not compromised by the process and by the fact that the cost of the product was not three to four times higher than conventional semen. Accordingly, a semen product that produces a gender bias in the offspring and at the same time does not have a huge impact on semen fertility would quickly establish a foothold in the market. The economic benefits of livestock sex pre-selection, in particular, are unequivocal. There is a high commercial need for a gender-biased product that does not impact greatly on semen fertility and also for a method to produce such semen that does not require expensive equipment, thereby resulting in semen being produced at lower cost.

[0006] The present inventors have found that through the use of a particular density gradient, and in some cases by maintaining the solution within a particular pH range, semen having an acceptable viability can be obtained.

[0007] The present method provides for the efficient use of the semen ejaculate in that it produces less sperm damage and consequently, higher semen fertility at lower equipment and operational cost. The present protocol aims to ameliorate the problems encountered in using the current flow cytometry-based method in the provision of a cost-effective and efficient method of separation of X- and Y-chromosome-bearing spermatozoa of cattle (Bos taurus and Bos indicus).

Summary of the Invention

[0008] The present inventors have developed a medium that allows fractionation of semen into two distinct fractions, wherein each fraction contains semen having a desirable viability. The preferred embodiment of the present invention is directed to the fractionation of raw bull semen; however, the invention is not to be taken to be restricted to cattle and is applicable to all mammals.

[0009] The raw cattle semen is preferably collected using the artificial vagina method from dairy bulls maintained by the Applicant. The quality of the raw semen is preferably assessed using computer-assisted semen analysis (IVOS-IT; Hamilton Thome Biosciences) and various other standard staining and microscopy techniques.

[0010] According to a first aspect of the present invention, there is provided a method of fractionating semen into X- and Y-chromosome-bearing spermatozoa comprising: layering an aliquot of semen on a disaccharide medium prepared as a density gradient having a concentration range of less than 15% w/v, wherein the pH range of the solution is maintained in the range of from 5.5 to 9.0; centrifuging the medium to obtain at least two distinct fractions of semen, a first contained in a lower, denser layer and a second contained in at least one upper, lighter layer, wherein the X-chromosome-bearing spermatozoa are enriched in the first fraction and the Y -chromosome-bearing spemiatozoa are enriched in the second fraction; and separating the upper layer from the lower layer. The density gradient preferably has a concentration range of from 3% to 14% w/v (i.e. g/lOOmL), and more preferably, from 6% and 14% w/v. Most preferably, the disaccharide medium comprises sucrose.

[001 1] In another preferred embodiment, the medium further comprises at least one buffer/s and/or at least one other semen extender additive/s. In a preferred embodiment, the buffer comprises tri-sodium citrate. Most preferably, the medium further comprises at least one commercially available animal protein-free semen extender/s, which is/are used to further increase the density of a solution. In a preferred embodiment, the semen extender is AndroMed \

[0012] In one preferred embodiment, the density gradient is constructed as a two-layer density gradient, wherein the pH of the lower, denser layer of the medium is preferably in the range of from 5.5 to 6.0, wherein the X-chromosome-bearing spermatozoa are attracted thereinto, and the pH of the upper, lighter layer of the medium is preferably in the range of from 8.5 to 9, wherein the Y-chromosome-bearing spermatozoa are attracted thereinto.

[0013] In another more preferred embodiment, the density gradients are constructed as three-layer density gradients, wherein the at least one upper, lighter layer comprises an intermediate, denser layer and an uppermost, lightest layer on top of the intermediate layer. Further more preferably, if the concentration range of the density gradient is from 6% to 14% w/v, the lowest layer has an acidic pH of from 5.5 to 6.0, the intermediate layer has a pH from above 6 to about 9 and the uppermost layer has an alkaline pH up to about 9. More preferably, the X-chromosome bearing spermatozoa are collected in the form of a pellet at the bottom of the lowest layer, and the Y-chromosome bearing spermatozoa are collected in the form of a pellet at the interface between the intermediate layer and the lowest layer positioned therebeneath. It is further preferred that the cattle semen is fractionated whereby the pellets formed are of substantially equal size. [0014] Further more preferably, if the concentration range of the density gradient is from 3% to less than 6% w/v, the lowest, densest layer has an acidic pH of from 5.5 to 6.7 and most preferably, a pH of 6.7.

[0015] In a most preferred embodiment, the pellets obtained after centrifugation are further washed with semen extender either by centrifugation and/or by sonication, and/or the dead spermatozoa are separated from the live spermatozoa in order to further improve the quality of fractionated semen.

[0016] According to another aspect of the invention, there may be provided a disaccharide medium prepared as a density gradient having a concentration range of from 3% to 14% w/v, wherein the pH range of the solution is maintained in the range of from 5.5 to 9.0, for use in the fractionation of semen into X-chromosome-bearing spermatozoa and Y-chromosome-bearing spermatozoa.

[0017] According to yet a further aspect of the present invention, there may be provided a method of sex pre-selection in a mammal, comprising the following steps: fractionating semen into X- and Y-chromosome-bearing spennatozoa according to the method of the present invention; collecting the X-chromosome-bearing spennatozoa from the lowest, densest layer and inseminating a female mammal with said spennatozoa, wherein the number of female progeny is increased; or collecting the Y-chromosome-bearing spermatozoa from the uppermost, lightest layer, whether that be from the uppermost, lightest layer for a two- layer density gradient, or from the interface between the intermediate layer and the lowest layer positioned therebeneath for a three-layer density gradient, and inseminating a female mammal with said spermatozoa, wherein the number of male progeny is increased.

[0018] Thus, the selective insemination step of dairy cows with the extended dairy bull semen, which has been processed with the fraction of semen enriched in

X-chromosome-bearing spennatozoa, may provide a cost-effective method for having greater numbers of heifer births in the dairy industry. The benefits offered to the dairy industry of being able to selectively produce a source of lactating cows are clear. Similarly, the selective insemination of beef cows with the extended beef bull semen, which has been processed with the fraction of semen enriched in Y-chromosome-bearing spermatozoa may provide a cost-effective method for having an increased number of male calf births in the beef industry, where bulls have an advantage of beef production. Description of the Figures

[0019] In order that the present invention may be readily understood and put into practical effect, reference will now be made to the accompanying illustrations, wherein like reference numerals refer to like features:

Figure 1 shows a reference slide;

Figure 2 shows the distinction between the lower, denser layer and the upper, lighter layer following layering, as indicated by the arrow;

Figure 3 shows an aliquot of the semen sample of a Holstein Friesian bull, as indicated by the arrow, layered on the top of the upper, lighter layer;

Figure 4 shows the centrifuge tube revealing two separate fractions post-centrifugation of almost equal volume of semen one in the upper, lighter layer and another in the lower, denser layer;

Figure 5 shows a slide of the pellet collected in lower, denser layer post-centrifugation in accordance with the present invention, wherein the circles identify X-chromosomes;

Figure 6 shows two separate fractions of semen, one in the upper, lighter layer and another in the lower, denser layer post-centrifugation according to the present invention;

Figure 7 shows the semen fractionated into two visible pellets;

Figure 8 shows the semen fractionated into two visible pellets of almost equal size, one in the upper, lighter layer and one in the lower, denser layer;

Figures 9 and 10 show a consistent semen fractionation into pellets using a three-layer density gradient;

Figure 1 1 shows the results of chromosomal staining using the aceto-orcein method on the raw semen, wherein the circles identify X-chromosomes; and

Figure 12 shows the increased population of X-chromosome-bearing spermatozoa, shown in circles, in the fractionated semen pellet having used the three-layer density gradient. Description of the Preferred Embodiment

[0020] The raw semen is preferably collected using artificial vagina method from dairy and beef bulls maintained by the present Applicant. The quality of raw semen was assessed using computer assisted semen analysis (IVOS-II; Hamilton Thorne Biosciences).

Preparation of Density Gradients

[0021 ] Density gradients were prepared using sucrose as the raw material in various concentrations ranging from 6% to 45% w/v. An aliquot of semen sample was stained for the chromosomal staining using the aceto-orcein staining method. The slide was examined under microscopic magnification of lOOOx using a Leitz Dialus 22 microscope for use as a reference slide, as shown in Figure 1.

[0022] A two-layer density gradient was initially prepared by layering a denser layer of 35% w/v at the bottom of the centrifugation tube, with the lighter layer 15% w/v layered on top of this. These concentrations were used in a previous study (Kanesharatnam et ai, 2012; International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 2, No. 3, May 2012, pp. 203- 206). An aliquot of the semen sample of a Holstein Friesian bull was layered on the top of the lighter layer, as shown in Figure 3, and a suitable number of such centrifugation tubes were prepared to make a balanced load in the centrifuge (Biofuge Primo, Heraeus Instruments) and the tubes centrifuged at speed-time combinations, ranging from 300 for 15 minutes to 900 ^ for 10 minutes. Post-centrifugation evaluation of these centrifuge tubes revealed two separate fractions of almost equal volume of semen that is one in the upper, lighter layer and another in the lower, denser layer, as shown in Figure 4.

[0023] These semen fractions were accordingly collected and put into separate 1.5 mL centrifugation tubes and centrifuged at 4000 rpm for 5 minutes (Biofuge Primo, Heraeus Instruments). The supernatant from each tube was then discarded and the pellet was carefully evaluated for:

(1) fractionation of spermatozoa using chromosomal staining by the aceto-orcein method; and

(2) semen quality using computer-assisted semen analysis (IVOS-II; Hamilton Thorne Biosciences). [0024] The slide for chromosomal staining from an aliquot of semen sample fractionated in 35% was examined under microscopic magnification of lOOOx using Leitz Dialus 22 microscope and an image was recorded, as shown in Figure 5. This was compared with the reference image of Figure 1 . It was observed that the pattern of chromosomal appearance in this image was different to the reference slide. A disproportionate number of X-chromosomes in comparison to the reference slide was observed. An altered chromosomal pattern between the reference image of Figure 1 without fractionation and the image obtained after semen fractionation was observed, which is a result of the altered population of spermatozoa after fractionation.

[0025] However, in the present experiments using these concentrations and a two-layer gradient, the inventors observed that the spermatozoa could not survive post-centrifugation in the lower, denser layer (concentration 35%) and the semen viability was severely reduced in the upper, lighter layer (concentration 15%), such that it was below the acceptable limit. By virtue of the physico-chemical properties of sucrose solutions, such as high osmolality and viscosity at such concentrations (Kanesharatnam et aL, 2012), these solutions might have resulted in the deleterious effects on spermatozoa. Therefore, the inventors decided not to use such high concentrations of sucrose for density centrifugation of spermatozoa in the present research. Hence, different concentrations of the sucrose media were then employed to avoid the reduction in the quality of the semen. Accordingly, experiments were undertaken using lower concentrations, some of which are shown in Table 1 , which suggest that semen quality was not compromised at lower concentrations. These concentrations were then used as indices to prepare density gradients to examine the efficacy of the media for semen fractionation.

Effect of some concentration of sucrose media on semen quality evaluated using computer assisted semen analysis (IVOS-II; Hamilton Thorne Biosciences)

Path velocity VAP (μηι/sec) 85.8 88.9 83.9 97.7 108.9

Progressive velocity VSL ^m/sec) 67.3 69.4 66.7 69.8 81.1

Track speed VCL (μηι/sec) 200.2 183.8 170.4 192 194.2

Straightness (%) 78 75 77 69 71

Linearity (%) 34 38 40 37 41

[0026] The aliquot of semen sample (Ι ΟΟμί) was layered on the top of upper, lighter layer as described above and the tubes were then centrifuged. Post-centrifugation examination revealed two separate fractions of semen, that is, one in the upper, lighter layer and another in the lower, denser layer, as shown in Figure 6.

Comparative Example using monosaccharide instead of disaccharide

[0027] In a second experiment, the effect of using monosaccharide media in the density gradient on cattle semen was examined. Fructose solution was prepared in concentrations ranging from 2% to 25% w/v. A density gradient was prepared using a 25% fructose solution as the lower layer and a 10% fructose solution as the upper layer. In another density gradient design, a 12% fructose solution was used as the upper layer instead of a 10% solution. An aliquot of semen sample (200μί) was layered on the upper layer in each case. Samples of each of these were prepared in different centrifuge tubes as described above and were then centrifuged at various time-speed combinations ranging from 200 g for 4 minutes to 400 g for 6 minutes. The semen was then fractionated in two visible pellets as shown in Figure 7. However, the quality of the semen was reduced below acceptable limits. Despite using lower concentrations of fructose media, this quality could not be improved.

Experiments to refine density gradient medium

[0028] Experiments were then undertaken to refine and modify the disaccharide and in particular, sucrose, used as a density gradient medium for the fractionation of cattle semen.

[0029] The concentrations of sucrose ranging from 6% to 18% w/v were prepared in tri-sodium citrate and in commercially available animal protein-free semen extender for use as density gradients. The two-layer density gradients were prepared in various combinations by using different concentration of the media prepared either in the tri -sodium citrate, or in the commercially available animal protein-free semen extender. This was achieved by layering the denser concentrations of from 14% to 18% w/v in the lower layer and lighter concentrations of from 7% to 12% w/v in the upper layer in a number of different centrifugation tubes. An aliquot of semen (400μί) was layered on the top of the upper layer in each tube. These tubes were centrifuged at various speed-time combinations ranging from 400 g for 9 minutes to 700 g for 6 minutes. The semen was separated into two visible fractions, preferably pellets, of almost equal size, that is, one in the lower layer and other at the interface between the two layers, as is shown in Figure 8.

[0030] The results of semen fractionation were consistent when density gradients with concentrations of from 14% to 16% w/v were layered in the lower layer and then concentrations of from 10% to 12% w/v were layered in the upper layer. During the above series of experiments, it was observed that media prepared in commercially available animal protein-free semen extender resulted in increased semen quality compared to a medium which was prepared in tri-sodium citrate. It was found that using two different density gradients prepared in different solutions, that is, one in semen extender and the other in tri-sodium citrate, can be used as viable density gradients, wherein the semen quality was found to be consistently good when the gradient was prepared with the commercially available animal protein-free semen extender.

[0031 ] Further, it was found that the preparation of three-layer density gradients was preferable to preparing two-layer density gradients. Accordingly, further experiments were undertaken by preparing three-layer density gradients in the order of having the densest medium at the bottom of the tube, the intermediate density on top of the densest medium and the lightest density in the uppermost layer, which resulted in a simpler method of recovering semen fractions from the intermediate layer and the lowest, densest layer being developed. An aliquot of semen (600μί to lmL) was layered on top of the uppermost layer. The tubes were centrifuged at various speed-time combinations ranging from 400 g for 9 minutes to 700 g for 6 minutes. A consistent semen fractionation was observed in all the experiments with three-layer density gradient design, as is shown in Figures 9 and 10. In addition, the results illustrating that the quality of semen obtained as a pellet from the lowest, densest layer was on the relatively higher acceptable scale compared to previous density gradient experiments of the current invention as shown in Table 2.

Table 2: Semen quality parameters of pellet fractionated in lowest, densest layer using sucrose as fractionation media, evaluated using computer assisted semen analysis (IVOS-II; Hamilton Thorne Biosciences)

[0032] Based on the experience of the inventors, the pellets obtained after centrifugation were washed with commercially available animal protein-free semen extender either by centrifugation and/or or by sonication in order to further improve the quality of fractionated semen. This resulted in marked improvement in the quality of fractionated semen as is shown in Table 3.

Table 3: Semen quality parameters post-washing of pellets fractionated in the lowest, densest layer using sucrose as fractionation media, evaluated using computer assisted semen analysis (IVOS-II; Hamilton Thorne Biosciences)

Straightness (%) 83

Linearity (%) 37

[0033] Consequently, chromosomal staining was performed using the aceto-orcein method on the raw semen as shown in Figure 1 1 and on the semen pellet obtained in the lowest, densest layer of the three-layer density gradient, as shown in Figure 12.

[0034] As shown in these Figures, the marked differences in the type of chromosomes seen in the raw semen, as opposed to the fractionated semen pellet obtained in the lowest, densest layer, clearly indicated an increased population of X-chromosomes in the latter. It can be concluded that the semen pellet separated in the lowest layer was enriched in the population of X-chromosome bearing-spermatozoa.

[0035] In yet a further experiment, the inventors prepared a three-layer density gradient in the order of having the densest medium at the bottom, the intermediate density on top of the densest medium and the lightest density in the uppermost layer. A falcon tube was pre- warmed to 34°C to 35°C. The concentration and semen quality parameters of raw semen obtained from a Holstein Friesian dairy bull using IVOS-II (Hamilton Thorne Biosciences; USA) was evaluated to ensure that the semen was of high acceptable quality to proceed with fractionation. A 3ml aliquot of a 0.15 mol/L sucrose solution, i.e. a 5% w/v solution, in commercially available protein-free semen extender with the pH adjusted to 6.7 in a 15 ml falcon tube was employed as the lowest layer. A 3 ml aliquot of 0.12 mol/L of this solution, i.e. a 4 % w/v solution, was employed on top of the lowest layer, thereby constituting the intermediate layer. Thereafter, a 3 ml aliquot of a 0.09 mol/L of the solution, i.e. a 3 % w/v solution, was placed on top of the intermediate layer, thereby constituting the uppermost layer. Finally, a 1.4 ml aliquot of raw semen of the dairy bull (Holstein Friesian) was placed on top of the uppermost layer. The falcon tube was centrifuged at 500 g for 5 minutes and then the tube removed from the centrifuge. The semen was observed as having been separated into two distinct pellets, whereupon the supernatant was decanted out of the tube. The pellet, which fonned at the bottom of the falcon tube, was transferred into another falcon tube and the concentration and semen quality parameters of the raw semen obtained from a Holstein Friesian bull using IVOS-II evaluated to ensure that the semen so fractionated was of acceptable quality to proceed with further extension. The semen was extended to final dilution as per standard laboratory practices. The semen straws were filled and sealed using the above extended semen as per standard procedures.

Effect of pH

[0036] Simultaneous experiments were also undertaken to identify the range of pH that would not result in any appreciable reduction of semen quality. In preliminary studies, the present inventors found that processing semen in media with an acidic pH of up to 5.5 and an alkaline pH of up to 9 did not result in any significant reduction in the semen quality parameters that would disqualify semen on a quality evaluation scale. These findings indicated the range at which the separation potential of pH can be used in the media of the invention to fractionate cattle semen.

Results

[0037] The results of the trials conducted are presented in Tables 4, 5 and 6 below. In the trials, the results of which are presented in Tables 4 and 5, cows were artificially impregnated with frozen semen, which had been fractionated according to the invention. In the trial, the results of which are presented in Table 6, cows were artificially impregnated with fresh semen, which had been fractionated according to the invention. In each case, the fraction that collected in the lowest layer was used to inseminate the cows.

[0038] In this first trial, of the twenty-two (22) cows that were impregnated, sixteen (16) cows were diagnosed as being pregnant. Ultrasound scans were initially conducted on the sixteen (16) pregnant cows, which showed that of the sixteen (16) pregnancies, ten (10) cows were carrying heifers and six (6) were shown to be carrying bull calves. These results were confirmed when the progeny were actually born. This represents a 62.5% bias towards producing heifers.

Table 4: Results of insemination of cows with frozen sperm fractionated according to the present invention

1 10 Female Female

2 10 Female Female

3 10 Female Female

4 10 Female Female

5 10 Female Female

6 9 Female Female

7 10 Female Female

8 10 Female Female

9 10 Female Female

10 10 Female Female

1 1 10 Male Male

12 1 1 Male Male

13 10 Male Male

14 10 Male Male

15 10 Male Male

16 1 1 Male Male

[0039] In this second trial, nineteen (19) cows were impregnated and eleven (1 1 ) cows were initially diagnosed as being pregnant. Ultrasound scans were conducted on the pregnant cows and the results showed that seven (7) out of the eleven (1 1) cows were carrying heifers and four (4) were carrying bull calves. These results were all confirmed when the progeny were actually born. This represents a 63.6% bias towards producing heifers.

Table 5: Results of insemination of cows with frozen sperm fractionated according to the present invention

2 10 Female Female

3 10 Female Female

4 10 Female Female

5 10 Female Female

6 10 Female Female

7 10 Female Female

8 10 Male Male

9 10 Male Male

10 10 Male Male

1 1 10 Male Male

[0040] In this third trial, fifty-three (53) carry over cows were impregnated with fresh sperm and sixteen (16) of them were diagnosed as being pregnant. One of these pregnant cows died before giving birth, leaving fifteen (15) cows remaining in the trial. The final results were that out of fifteen (15) pregnancies, eleven (1 1) heifers and four (4) bull calves were born, showing a bias of 73.3% towards producing heifers.

Table 6: Results of insemination of carry over cows with fresh sperm fractionated according to the present invention

8 Male (?) Female

9 Female Female

10 Female Male

1 1 Female Female

12 Female Female

13 Female Female

14 Female Female

15 Female Female

16 Male Male

[0041 ] Throughout the specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

[0042] Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.