A re-diluted bulk frozen semen (RDF) technology developed by us and used commercially involves the following stages: Ejaculate collected and basic QA applied.

One variant separates out a majority of natural semen diluent (seminal plasma) from sperm in semen and adds a suitable cryoprotective diluent (based on for example: Glycerol, Dimethyl Sulphoxide, Ethylene Glycol, Trehalose, Sucrose, Glucose) bringing the semen to an appropriate sperm concentration.

Another variant dilutes the sperm in semen to an appropriate concentration in a suitable cryoprotective diluent (e. g.; glycerol or a glycerol based diluent).

Sperm cells, now at approximately 25% of their initial concentration, are frozen (50-600 million sperm/ml).

When using the semen stored this way, there is a need to thaw and then add the bulk frozen semen to a semen diluent (such as CAPROGEN semen diluent). That mix is then loaded into straws for use as if fresh semen: (i. e. to be used within 3 days and not refreezed).

CAPROGEN semen diluent cannot be frozen with the semen since we have found it will not maintain viable sperm.

BACKGROUND ART It is an object therefore of the present invention to provide viable semen straws, semen containing containers, viable insemination straws, related uses and methods of use as well as procedures for providing such straws or containers (including equipment therefor) which will at least provide an alternative to those known. More particularly it is an object in some embodiments of the present invention to provide a capability whereby a concentrated semen to be cryogenically stored for later use can be used without any additional addition.

SUMMARY OF THE INVENTION In a first aspect the present invention consists in a viable semen straw in cryogenic storage, said straw being characterised in that there is a partitioned content of (i) a semen content capable of cryogenic storage, and (ii) a semen diluent content capable of cryogenic storage, andfurther characterised in that only upon thawing from cryogenic conditions can the partitioned content mingle to any significant extent.

As used herein the term"straw"means or includes any suitable container suitable for both in the field use for insemination purposes and for cryogenic storage, the container preferably being substantially tube-like (any cross-section and irrespective of whether of variable cross-section) with a capability (with or without opening or breaking) of allowing the contents to be discharged or to discharge. See for example US Patent Nos. 4, 821, 490, 5,190, 880, 5, 283, 170 and 6,305, 585 the full contents of which are here hereby incorporated.

Preferably the straw is a tube of an elongate open ended form.

As used herein the term"semen"is as used in the insemination art i. e. it can be natural, concentrated or diluted semen. Reference to viability is of the sperm content thereof as also is concentration.

As used herein"diluent"in respect of use with semen prior to cryogenic freezing includes any appropriate diluent (e. g.; not contraindicated for cryogenic freezing with semen) and in respect of post thaw use includes any appropriate system (e. g.; extender) adapted to support the semen (e. g.; to enhance viability).

As used herein the term"viable semen straw"means or includes such a"straw" including a content viable as intended to be used for insemination purposes in fertilising a female's egg.

As used herein the term"and/or"means"and"or"or"both.

Preferably said mingling requires shaking of the straw.

Preferably said partitioning is partitioning by a gas interposed between the (i) and (ii) contents.

Preferably the species of the animal from which semen is derived can be selected from any of many species typified (but not limited to) the bovine, ovine, equine, porcine,

hominid (humans, apes and monkeys), camelid, caprine, lapine, rodent or like mammalian species.

Preferably said semen content (when of the bovine species) (i) is such that when mingled with the diluent content (ii) the overall semen concentration is below the current industry standard for bovine AI, possibly by at least a factor of a half.

Preferably said semen content is modified, i. e. by removal of some of the natural semen diluent or by dilution to the appropriate concentration in an appropriate diluent, or both. Glycerol is one such appropriate diluent. Others include those earlier listed.

In a further aspect the present invention consists in a viable semen straw capable of cryogenic storage, said straw being characterised in that there is a (preferably gas) partitioned content of (i) a semen content capable of cryogenic storage, and (ii) a semen diluent content capable of cryogenic storage.

Preferably said straw can be cryogenically stored without the partitioned content having mingled to any significant extent.

Preferably said straw after thawing can be shaken to effect sufficient mingling of contents (i) and (ii) to provide a viable semen content suitable for subsequent insemination.

Preferably said semen content is modified, i. e. by removal of some of the natural semen diluent or by dilution to the appropriate concentration in an appropriate diluent, or both.

Preferably the species of the animal from which semen is derived can be selected from any of many species typified (but not limited to) the bovine, ovine, equine, porcine, hominid (humans, apes and monkeys), camelid, caprine, lapine, rodent or like mammalian species.

In yet a further aspect the present invention consists in a viable semen straw in or capable of cryogenic storage, said straw being characterised in that there is a gas partitioned content of (i) a semen content capable of cryogenic storage, and (ii) a semen diluent content capable of cryogenic storage wherein contents (i) and (ii) can be caused to intermingle, and wherein said semen content is of a fraction of normal semen.

Preferably said fraction is modified insofar as sperm per volume of the resultant semen content is concerned.

Preferably the species of the animal from which semen is derived can be selected from any of many species typified (but not limited to) the bovine, ovine, equine, porcine, hominid (humans, apes and/or monkeys), camelid, caprine, lapine, rodent or like mammalian species.

In another aspect the present invention consists in a semen containing container in or capable of cryogenic storage adapted to make the semen available for insemination purposes (to a egg or to the female animal itself), characterised in that there is a partitioned content of (i) a semen content capable of cryogenic storage, and (ii) a semen diluent content capable of cryogenic storage.

Preferably the partitioning is with a gas.

Preferably the semen content is semen at the requisite concentration.

Preferably the species of the animal from which semen is derived can be selected from any of many species typified (but not limited to) the bovine, ovine, equine, porcine, hominid (humans, apes and/or monkeys), camelid, caprine, lapine, rodent or like mammalian species.

In yet another aspect the present invention consists in a viable insemination straw in cryogenic storage, said straw being characterised in that there is a distinct semen content capable of cryogenic storage and a distinct semen diluent content capable of cryogenic storage, and further characterised in that prior to exposure to cryogenic conditions the distinct contents have not mingled to any significant extent, and further characterised in that an active input (e. g. shaking, etc. ) can, after thawing, lead to intermingling or mixing of the distinct contents in the straw.

Preferably said semen content is modified, i. e. by removal of some of the natural semen diluent or dilution of some or all of the natural semen diluent.

Preferably the species of the animal from which semen is derived can be selected from any of many species typified (but not limited to) the bovine, ovine, equine, porcine, hominid (humans, apes and/or monkeys), camelid, caprine, lapine, rodent or like mammalian species.

In yet another aspect the present invention consists in a method of providing a viable semen straw in or capable of cryogenic storage, said method comprising or including the steps of, in any order

inserting in the straw a semen content capable of cryogenic storage, and inserting in the straw a semen diluent content capable of cryogenic storage, wherein the insertions is without any substantial mingling of one content with the other.

Preferably said semen content is modified, i. e. by removal of some of the natural semen diluent or by dilution to the appropriate concentration in an appropriate diluent, or both.

Preferably the species of the animal from which semen is derived can be selected from any of many species typified (but not limited to) the bovine, ovine, equine, porcine, hominid (humans, apes and/or monkeys), camelid, caprine, lapine, rodent or like mammalian species.

Preferably the content (s) of any of the foregoing aspects of the present invention is (or are) of a kind (or kinds) substantially as hereafter exemplified.

In yet another aspect the present invention consists in a viable semen straw in or capable of cryogenic storage, said straw provided by a method as aforesaid.

In another aspect the invention consists in the use of a viable semen straw or a viable insemination straw of the present invention for insemination purposes of a female or an egg.

Preferably the use for insemination purposes is after intermingling or intermixing of the contents of the straw.

Preferably said intermingling or intermixing (it not mattering whether or not there is mutuality in movement or not) occurs after thawing from cryogenic storage.

Preferably the use occurs sometime after cryogenic thawing, e. g. possibly without cryogenic storage between some cryogenic storage depot and the farm. Such straws however may be held in cool or ambient conditions between such thawing and actual insemination use.

Preferably the species of animal from which the semen was derived or any of the forms of viable semen straw or viable insemination straw in accordance with the present invention and the related methods etc. of use is that of the bovine, ovine, equine, porcine, hominid (humans and/or monkeys), camelid, caprine, lapine, rodent or like mammalian species.

In one embodiment the semen is of the bovine species.

In still a further aspect the present invention consists in apparatus suitable for performing a method in accordance with the present invention which provides a viable semen straw, said apparatus including or comprising (optionally) apparatus to modify semen, apparatus to insert a semen content into a straw, and apparatus (whether the same as for the semen, or different or another) to insert a semen diluent into the straw prior to, simultaneously with and/or subsequent to the insertion of the semen into the straw.

The insertion and sealing can be by any appropriate means including those discussed in the aforesaid US Patents.

Preferably said apparatus has means to cause or to allow the location of a gas (e. g. nitrogen or air) partition between the two contents (e. g. said insertion apparatus may trap a gas between the inserted materials).

Forms of the invention will be described in more detail hereinafter.

In a further aspect the present invention consists in a method of preparing an insemination straw for cryogenic storage, said method at least including the steps of inserting discretely two contents into the straw, one content being of semen (concentrated or otherwise) and the other being of a semen diluent, such insertions leaving the distinct contents separated or partitioned.

In another aspect the invention consists in apparatus for performing a method of preparing an insemination straw for cryogenic storage, said method at least including the steps of inserting discretely two contents into the straw, one content being of semen (concentrated or otherwise) and the other being of a semen diluent, such insertions leaving the distinct contents separated or partitioned, said apparatus comprising or including a straw carrying drum, wheel, or other conveyor ("conveyor") adapted to receive and index single or groups of straws ("straw (s) ") straws at a loading zone, move continuously or intermittently carrying the indexed straw (s), and to discharge them as filled straw (s) at a discharging zone distinct from the loading zone, a first filling nozzle or set of filling nozzles ("first filling nozzle (s) ") for indexing a<BR> first end or first ends ("first end (s) ") of a straw or straws ("straw (s) ") carried to a first filling zone between the loading and discharging zones,

a first vacuum nozzle or set of vacuum nozzles ("first vacuum nozzle (s) ") for<BR> indexing to a second end or second ends ("second end (s) ) of the straw (s) being indexed by the first filling nozzle (s), <BR> <BR> a second filling nozzle or set of filling nozzles ("second filling nozzle (s) ") for<BR> indexing to first end (s) of a straw or straws ("straw (s) ") carried to a second filling zone between the loading and discharging zones, the second filling zone being closer to the discharging zone than is the first filling zone, a second vacuum nozzle or set of vacuum nozzles ("second vacuum nozzle (s) ") for indexing to the second end (s) of the straw (s) being indexed by the second filling nozzle (s), wherein the first filling nozzle (s) and first vacuum nozzle (s) co-act to load a first content into an indexed straw and the second filling nozzle (s) and second vacuum nozzle (s) co-act to load the other content into each straw.

Preferably one or both of said first and second vacuum nozzle (s) is allowed to draw in a partitioning gas between the loadings of the contents to be separated or partitioned.

Preferably said conveyor is a drum capable of receiving single or multiple straws at the loading zone and to present the ends thereof on an axis longitudinally of each straw that is parallel to the rotational axis of the drum As used herein the term"straw"and any variations thereof such as"viable semen straw","insemination straw", etc. preferably refers to a container such as a tube having a passageway in which semen might be stored in a cryogenic liquid such as liquid nitrogen.

Preferably such straws prior to filling are open ended and are of a constant passageway section (usually circular). Without being restricted as to the type of straw that may be utilised, preferably such straw, whether of glass or a suitable plastics or other material might have an internal diameter of, for example, about 1. 5mm and a length of, for example, 135mm. Such dimensions and configurations of a suitable container within the ambit of the term"straw"are not to be considered limiting as to the term.

Preferably the viable semen straw is capable of being used to inseminate an appropriate recipient female animal or eggs of such an appropriate animal.

The present invention recognises as an answer to the abovementioned problems the insertion of both concentrated semen and a semen diluent (such as CAPROGEN@) semen diluent, milk, egg yolk, etc. within the same straw separated by a suitable partition (e. g. a gas bubble partition). Thus it can be stored frozen (since at least CAPROGEINTO as a semen diluent [and the others as well] is able to be frozen by itself) but once thawed the straw, if

shaken, can dislodge the gas bubble and get the appropriate mixing of (e. g. bovine) semen and CAPROGEN or other diluent for successful artificial insemination (Al). This could be used practically in the field by the artificial breeding (AB) technicians.

Sperm in the form of such post thaw mixed (concentrated) semen and semen diluent maintains its viability in the straw for an extended post thaw and post mix period so the straw of semen could be thawed at a central facility and distributed without the need for cryo-tanks and liquid nitrogen.

BRIEF DESCRIPTION OF THE DRAWINGS Preferred forms on the present invention will now be described with reference to the accompanying drawings in which, Figure 1 shows by a series of Figure 1A through 1D an empty straw having a porous seal (A) at the left hand end which hardens upon contact with liquid, the diluent (B) and gas bubble partition (C) being inserted into the straw (Figure 1B), the semen (D) being drawn into the straw behind the diluent and the gas bubble partition and finally (Figure 1D) the tube being sealed (at E) by a sonic sealer beyond a gas or air filled space after the semen (D) has been drawn in behind the gas bubble partition, Figure 2 shows a flow diagram from the bull to end usage that is generic of the various options discussed herein of the present invention, Figure 3 shows a first procedure for straw filling, Figure 4 shows a second procedure for straw filling, Figure 5 is an exploded view but showing the general assembly of preferred apparatus in accordance with the present invention, Figure 6 shows the assembly from one perspective but not showing covers, Figure 7 is a view of the same assembly of Figure 6 but from a different perspective, Figure 8 is a plan view of the apparatus of Figures 5 to 7 but showing the covers, Figure 9 is a side elevation view of the apparatus as shown in Figure 8, Figure 10 shows the section'AA'of the apparatus of Figures 8 and 9 in elevation, and Figure 11 shows the apparatus of Figures 5 to 10 but with one cover part removed and showing linkage to a control console.

DETAILED DESCRIPTION OF THE INVENTION The present invention is not species specific but preferred forms will be described primarily with respect to the bovine species but on the understanding that performance for viable thawed semen of other species (e. g. as disclosed by way of example) demonstrate the principles involved.

DILUTION METHOD ALTERNATIVE 1: (DIALYSIS SYSTEM) Bovine semen is diluted at 30°C into a primary diluent (Part A) to an appropriate sperm concentration between 50 and 600 million/ml. Nitrogen gas is injected into the head space of the vessel containing the primary dilution. The vessel is placed into a thermal buffer consisting of a water jacket of 200mls water at 30°C then placed into a 5°C refrigerated cabinet to cool for at least 30 minutes. After 30 minutes the primary is removed from the water jacket and allowed to equilibrate to the refrigerator temperature for another hour.

The semen and primary diluent admixture is then transferred to a pre-prepared dialysis sac (12kda mwt cut-off) which is tied at both ends. The sac is placed into a pre- prepared vessel containing an appropriate admixture of diluent parts B and C to achieve a final glycerol concentration of 8% in the entire dialysis containment. This is achieved by making a mixture of the following proportions: 50% part C plus a portion of part B equivalent to 50% minus the percentage of the final cylinder volume represented by the volume of the semen plus primary diluent admixture. When the sac is placed in this mixture it should displace sufficient fluid to make the entire contents of the vessel up to 100%.

Any head space in the dialysis vessel is filled with nitrogen gas. The mixture is dialysed at 5°C for 16 hours. The dialysed semen admixture is transferred to measuring vessel and the volume adjusted to make up any loss over the dialysis period. Adjustment is made with a 1: 1 mixture of parts A and C to maintain a glycerol concentration of 8%.

Dialysis method: (MacMillan, Shannon et al. 1978) (Shannon 1972) (Shannon and Vishwanath 1995) (Vishwanath, Pitt et al. 1996).

DILUTION METHOD ALTERNATIVE 2: (TRIS SYSTEM) : Bovine semen is diluted to an appropriate concentration between 50 and 600 million sperm/ml at 30°C into a primary diluent described in the reference below. The method is modified to include the addition of a peroxidase enzyme and the exclusion of oxygen by nitrogen gas bubbling. Nitrogen gas is injected into the head space of the vessel containing the primary dilution. The vessel is transferred to a refrigerated cabinet at 5°C to cool and equilibrate for 3 hours. <BR> <BR> <P>Foote, R. H. (1970). "Fertility of Bull Semen at High Extension Rates in Tris<BR> Buffered Extenders. "Journal of Dairy Science (53): 1475-1477.

By means of a suitable machine described in the second part of this application an appropriate volume (e. g. 12. 5111) of the semen admixture is placed into a 0. 25ml, a 0. 5ml or any other appropriate volume straw that already contains an appropriate volume (e. g.

200u. l) of CAPROGENO diluent or any other diluent that extends the life of semen at non- cryogenic temperatures. A gas partition or barrier is drawn into the straw between the semen containing portion and the ambient temperature preservation diluent portion.

The straw is sealed ultrasonically, by heat, or by means of a powder plug. The straw is then suspended at an appropriate height above the level of the liquid nitrogen in a static vapour freezer for an appropriate time then plunged into the liquid nitrogen to complete freezing or the straw is suspended in a controlled atmosphere freezing device and subjected to a programmed freezing regime deemed appropriate for the particular ejaculate.

The semen can then be maintained frozen indefinitely until required for use.

DILUTION METHOD ALTERNATIVE 3: Any other means of cryogenically preserving semen from any species that retains fertility of semen after freezing at cryogenic temperatures and subsequent thawing before insemination or any other means of achieving fertilisation (e. g. in vitro fertilisation).

THAWING PROCEDURE: Thawing of the individual semen dose is or can be achieved by plunging into ambient temperature water for 15 seconds. The straw is then dried and the ultrasonic seal is cut off with scissors before loading into the insemination device.

Upon thawing the artificial breeding technician shakes the straw to mix the two components before insemination into the animal.

Other procedures however can be used that achieve thaw without any substantial viability loss.

MACHINE DESCRIPTION: A preferred machine description will now be given with reference to Figures 5 through 11 in which the references depict the following: 1. Frame Assembly 2. Drum Assembly 3. Socket Head Cap Screw 4. Hopper Assembly 5. Socket Head Cap Screw 6. Vacuum Side Assembly 7. Injection Side Assembly 8. Straw 9. Ultrasonics 10. Tray 11. Side Cover 12. Side Cover Mirrored 13. Tray Guide 14. Inspection Hatch 15. Nut 16. Counter Sunk Cap Screw The machine consists of a hopper (part of the assembly 4) to contain approximately 750 straws. Straws are perturbated by a low frequency oscillating plate (part of the assembly 4) which is driven by a servomotor to ensure they feed freely and coherently. The straws are fed by gravity from the base of the hopper into a transport mechanism consisting of a slotted drum (part of the assembly 2) that rotates inside an enclosure.

The drum is driven via a belt by a stepper motor (part of the assembly 2). The drum rotates sufficiently to pick up straws in sets of one, two or three from the hopper. The straws are indexed into slots in the rotating drum and are transported around the drum enclosure to a point adjacent to a set of one, two, or three silicone gasketted filling nozzles (part of the assembly7). A machine vision system detects the presence or absence of a straw/s and communicates with the injection system to give an'inject or don't inject' condition. The filling nozzles are connected by a flexible, biocompatible tube to a reservoir of the ambient temperature preservation diluent. The nozzles engage the straws and a controlled vacuum is applied to the other (wadding plug) end by a similar set of nozzles (part of the assembly 6). An appropriate volume of CAPROGENS diluent (approximately 185 up or any other diluent that extends the life of semen at non-cryogenic temperatures is drawn into the straw to an appropriate distance from the inner end of the wadding plug or an appropriate distance from the opposite end of the straw.

The filling nozzles are timed to disengage from the straw slightly ahead of the vacuum nozzles so a vacuum is maintained in order to pull an appropriate volume (approximately 1 Ouo of ambient gas into the straw. This forms a gas partition between the diluent column and the subsequently injected semen column. The vacuum nozzles then disengage from the straws.

The set of one, two, or three straws is then transported by the rotation of the drum to the next set of filling nozzles (part of the assembly labelled 7). This second set of filling nozzles is connected by a flexible, biocompatible tube to a reservoir containing the diluted, processed semen of which an appropriate volume (approximately 18 ul) is injected by means of a peristaltic pump. A vacuum is applied by a second set of vacuum nozzles (part of the assembly labelled part No. 6 in drawing 86233-200). The vacuum draws the column of CAPROGEN diluent or any other diluent that extends the life of semen at non- cryogenic temperatures already present in the straw into the wadding and powdered Polyvinyl Acetate (PVA) or, any other powdered gelling material plug, thereby sealing it.

This action also draws the semen column further into the straw.

The placement of the semen plug is controlled by adjustment of the length of the filling nozzle and the timing of the injection into the straw. The injector nozzle places the semen appropriately to leave sufficient space in the end of the straw for the ultrasonic seal to be accommodated.

The set of one, two, or three straws is then transported by further rotation of the drum to an ultrasonic sealing unit 9 where the straws are sealed by ultrasonic welding. The welding head (emitter) is static while the welding plate (anvil) is reciprocated up to meet the straws and down to allow the passage of each set of straws and the ingress of the next set.

The straws are ejected by further rotation of the drum into a collection tray 10 to await freezing or, if not ultrasonically welded, to be heat-sealed or powder plug sealed.

CAPROGENt extender: (Shannon 1964; Shannon 1968) (Vishwanath, Pitt et al.

1996 ; Krzyzosiak, McMillan et al. 2000) (Shannon 1965) (Shannon and Curson 1972).

EXAMPLES BOVINE FIELD TRIAL Treatment Control Number of successes (e. g. pregnancies): 457 654 Total number of valid trial data 793 1,139 Proportions 57.6% 57.4% Actual Treatment-Control Proportion 0. 21 % Probability that treatment-control Is better or equal to tolerance Threshold of-5%: 98.9% Likelihood that true value = >-5% Versus true value <-5% i. e. 87.

Power of the trial (given expected T-C = 0%, a tolerance limit for T-C CI @-5%, and 1-tailed CI of 95%): 70.7% Probability that treatment is Equal to or better than Control : 53. 7% Likelihood that actual Treatment = > control Versus < control : 1.2

Confidence intervals for treatment-control result: 99% CI : tailed CI)-5. 11% to 6.10% i. e. 1% chance that true result is less than-5.11% 95% CI : (l-tailed CI)-3. 55% to 6.10% i. e. 5% change that true result is less than-3.55% 90% CI: (1-tailed CI)-2. 72% to 6.10% i. e. 10% chance that true result is less than-2.72% EVALUATION OF OTHER SPECIES Species of semen extracted from species other than bovine were then tested. Our minimum standard for bovine semen in like circumstances is 45% live and 7 motility.

In these in vitro examples semen was sourced from ABS in freezing diluent appropriate for each species. It was then processed in a 2-part straw using CAPROGENX as the secondary diluent. Thawed in 37°C water for 15 seconds, it was then shaken to mix and then evaluated as to percentage live sperm and motility in the resultant semen mix.

Ohrs % live motility/10 Boar 60 8 Buck 65 8 Ram 45 7 Horse 40 6 NOVEL ART AND USEFULNESS OF THE INVENTION The stepwise filling process and method for semen is new. The partitioned content is new. The incompatibility of the CAPROGEN° components with cryopreservation has been overcome by this two-stage method. The superior semen preservation properties of CAPROGEN (see reference Shannon P (1965) hereafter listed) are utilised without their deleterious effects at the freezing stage. Two-stage filling is novel for semen preservation.

The timed application of the vacuum used to pull the diluent column and injection to place the semen column into the straw is new. Present processes use crude control via pinch valves. The introduction of an air plug between the two diluent components by means of the timing of the vacuum and injection application is new.

The inclusion of a separator between the semen and diluent columns is also a novel element.

Semen frozen and stored in this manner will be able to be used up to 72 hours after thawing. Viability is maintained inside the straw in contrast to the standard frozen semen treatments, which must be inseminated within minutes of thawing. This will allow the use of a central storage and thawing facility thereby eliminating the need for individual inseminating technicians to carry a cryogenic container for frozen semen. This is a novel concept that cuts costs by limiting capital expenditure to only the liquid nitrogen storage facilities at the central site and by cutting overheads in consumables, namely, liquid nitrogen.

BIBLIOGRAPHY : Krzyzosiak, J. , G. McMillan, et al. (2000)."Protein Tyrosine Phosphorylation During Prolonged In Vitro Incubation of Ejaculated Bovine Spermatozoa Is Regulated by the Oxidative State of the Medium."Biology of Reproduction 62: 1615-1623.

MacMillan, K. L. , P. Shannon, et al. (1978)."The redilution of deep frozen semen." Animal Production 26: 67-73. w Shannon, P. (1964). "The effect of diluents containing glycine, and glycine and<BR> glycerol, on the fertility of diluted bovine semen. "New Zealand Journal of Agricultural Research 7: 357-363. g Shannon, P. (1965). "Contribution of seminal plasma, sperm numbers and gas phase to the dilution effect of bovine spermatozoa."Journal of Dairy Science 48: 1357-1361.

Shannon, P. (1968). "Advances in semen dilution."Proceedings of New Zealand society of animal production 28: 23-31.

Shannon, P. (1972). "The diatube method of freezing semen for use as a rediluted deep freeze."Proceedings Vin International congress Animal production and artificial insemination, Munich 4: 1360-1362.

Shannon, P. and B. Curson (1972). "Toxic effect and action of dead sperm on<BR> diluted bovine semen. "Journal of Dairy Science 55 (5): 614-620.<BR> <P>Shannon, P. and R. Vishwanath (1995). "The effect of optimal and suboptimal concentrations of sperm on the fertility of fresh and frozen bovine semen and s theoretical model to explain fertility differences."Animal reproduction science 39: 1-10. <BR> <BR> <P>Vishwanath, R. , C. J. Pitt, et al. (1996)."Sperm numbers, semen age and fertility in fresh and frozen bovine semen."Proceedings of the New Zealand Society of Animal Production 56: 31-34.