DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
1. The Amendment filed February 17, 2026 in response to the Office Action of November 17, 2025, is acknowledged and has been entered. Claims 1-4, 18-25 are pending. Claims 5-17 are canceled. Claim 1 is amended. Claims 23-25 remain withdrawn as being drawn to non-elected Group II. Claims 1-4 and 18-22 are currently being examined.
Maintained Rejections
(with amendments addressed)
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
2. Claims 1-4 and 18-20 remain rejected under 35 U.S.C. §103 as being unpatentable over Durack et al. (International Patent Application Publication No. WO 2004/088283 A2) in view of Gomes-Alves et al. ((2014) Theriogen. 82: 469-474), Moreno et al. ((2013) J. Equine Vet. Sci. 33: 1068-1075) and Oh et al. (Korean Patent Application Publication No. KR20150003058A; Pub. Date: 2015-01-08; see English machine translation (EngMT) as NPL for page/para. numbers).
Regarding claim 1, pertaining to a method of reducing agglutination and improving health of sperm cells having an altered ratio of viable X and Y chromosome-bearing sperm cells,
Durack et al. shows methods relating to the collection and processing of animal semen, particularly processing semen in order to sort sperm cells according to a specified DNA characteristic (e.g., X/Y content to preselect the gender of offspring) (pg. 22, lines 2-4). Some embodiments of the described invention employ improved cryopreservation methods to reduce the time required for cryopreservation and/or to improve the health of the cryopreserved sperm (pg. 116, lines 13-16).
Further regarding claim 1, pertaining to staining a sperm cell sample with a staining media comprising a DNA selective dye, Durack et al. shows that the staining protocol that will be used for processing is selected by using various staining protocols to stain aliquots of the sample and then analyzing the sortability of each aliquot to identify a desired staining protocol for that particular sample. Staining according to the selected staining protocol is performed by adding a staining fluid containing a chemical dye (e.g., a DNA selective fluorescent dye) to each semen sample (pg. 22, lines 19-24). Uptake of dye by the sperm cells in the staining mixture is allowed to continue for a period of time sufficient to obtain the desired degree of DNA staining. That period is typically a period sufficient for the dye to bind to the DNA of the sperm cells such that X and Y chromosome-bearing sperm cells may be sorted based upon the differing and measurable fluorescence intensity between the two (pg. 29, lines 18-22).
Further regarding claim 1, pertaining to contacting the stained sperm cell sample with a sheath fluid in a flow path, Durack et al. shows that a sample is loaded into the sample introduction device of a flow cytometer. The sample fluid is introduced into the flow cytometer along with a sheath fluid. In addition to the sheath fluid, additives may also be added (pg. 22, lines 27-30). Improved orientation of particles may be obtained by altering the flow of the fluid stream through an orienting nozzle so that the core stream containing the particles to be oriented (e.g., sperm cells) is directed along a flow path (pg. 37, lines 22-26).
Further regarding claim 1, pertaining to altering a ratio of the viable X chromosome-bearing sperm cells to the viable Y chromosome-bearing sperm cells to form at least one altered sperm cell population, Durack et al. shows that X and Y chromosome-bearing sperm cells may be sorted based upon the differing and measurable fluorescence intensity between the two (pg. 29, lines 18-22). The width of the focused beam spot of the flow cytometer is, preferably, less than the chromatic DNA mass of the cell (pg. 51, lines 6-12). One (of two) intercepting devices for each cytometry unit can direct fluid into a common waste trough rather than a collection vessel for discarding sorted droplets that contain particles of little value (e.g., Y-chromosome bearing sperm cells for breeding dairy cows) (pg. 134, lines 10-13).
Further regarding claim 1, pertaining to collecting the altered sperm cell population in a collection media, Durack et al. shows that the sorted sperm cells are collected by the collection system of the flow cytometer, and they are added to a collection vessel that contains a collection fluid or cryoextender (pg. 22, lines 33-35).
Further regarding claim 1, pertaining to a collection media comprising egg yolk, egg yolk at a wt./vol. concentration of 10% or less, Durack et al. teaches that the sorted sperm are collected in a vessel that contains a collection fluid. The collection fluid may comprise a protein source (pg. 103, lines 28-33). If included, the protein source may be any protein source that does not interfere with the viability of the sperm cells and is compatible with the particular buffer or buffered solution being used. Examples of common protein sources include, minimally, egg yolk and egg yolk extract (pg. 104, lines 3-6). Durack et al. teaches that such proteins, if included in the collection fluid, may be used in a concentration of from about 1% (v/v) to about 30% (v/v) or 10% (v/v) (pg. 104, lines 6-8).
Durack et al. does not explicitly show: 1) reducing agglutination of sperm cells [Claim 1]; 2) a low-density lipoprotein additive; 3) the egg yolk is present in the collection media at a wt/vol concentration of about 0.1%; and 4) the low-density lipoprotein additive is present at a wt/vol concentration of about 2.5% [Amended Claim 1].
Gomes-Alves et al. shows a study in which different bovine and canine commercial semen extenders for the cryopreservation of brown bear ejaculates was evaluated. An extender (Test-Tris-fructose-egg yolk-glycerol (TTF-ULE/bear) that was developed for brown bears served as a control extender (pg. 469, Abstract [nexus to Durack et al.- sperm extenders/additives, egg yolk]).
Regarding claim 1, pertaining to reducing agglutination of sperm cells,
Gomes-Alves et al. shows the study as also evaluating the effect of semen collection directly into extender on sperm agglutination (pg. 469, Abstract). The Test-Tris-fructose (TTF) base extender was prepared. The TTF base solution was supplemented with 20% egg yolk and antibiotics. This constituted the control extender (TTF-egg yolk-glycerol [ULE]/bear. The agglutination handling extender (TTF-H) was prepared from the TTF base extender supplemented with 1% egg yolk (pg. 470, column 2, para. 2 [nexus to Durack et al.- egg yolk in sperm cell collection media]). Table 2 shows the assessment of various characteristics of brown bear sperm diluted in the various extender compositions compared (e.g., motility, viability, mitochondria potential), pre-freezing and post-thawing (pg. 471, column 2, last para.; and pg. 472, Table 2). All egg yolk-based extenders used in the described study have the same concentration of egg yolk (pg. 472, column 2, para. 2). Collection of ejaculates directly in TTF-H extender decreases sperm agglutination in fresh samples (pg. 473, column 1, para. 3).
Moreno et al. shows the advantages of low-density lipoproteins for the cryopreservation of equine semen (pg. 1068, Title). Egg yolk is a component of most so-called semen extenders, because it protects the sperm from thermal shock during freezing. The cryoprotective fraction of egg yolk is due to a low-density lipoprotein (LDL) component, which was isolated and purified (pg. 1069, column 1, para. 1 [nexus to Durack et al. and Gomes-Alves et al.- egg yolk to improve health of sperm cells, and to reduce agglutination, respectively]).
Regarding claim 1, pertaining to a low-density lipoprotein additive,
Moreno et al. shows a study to determine the best concentration of low-density lipoprotein (LDL) in semen extender to improve the percentage of motile spermatozoa in equine sperm after freezing and thawing in comparison with commercial extenders, including egg yolk. The best post-thaw motility results were obtained with the extenders made with 0.5%, 2%, and 3% LDL and with the control extender made with egg yolk; no significant difference was observed between these extenders (pg. 1068, Abstract).
Oh et al. provides information that would have motivated one of ordinary skill in the art of producing a semen or sperm extender containing egg yolk, as shown by Durack et al., to have added a low-density lipoprotein additive, as shown by Moreno et al., by way of addressing the limitations of claim 1.
Oh et al. shows a composition for sperm cryopreservation, including a low density lipoprotein (LDL) and an antioxidant, and a method for increasing activity of cryopreserved sperm (pg. 2, Abstract cont.). The low density lipoprotein (LDL) is capable of inhibiting sperm deactivation during cryopreservation (pg. 2, para. [0001] [nexus to Moreno et al.- semen extender comprising LDL; nexus to Durack et al. and Gomes-Alves et al.- semen extender compositions]). A freezing preservation liquid is described (pg. 9, para. [0023]).
Regarding claim 1, according to the invention described in Oh et al., the term "freezing preservation liquid" means a sperm cryopreservation formulation in a liquid mixture of the cryopreservation composition and diluent of the described invention. The diluent is not particularly limited thereto, but may be a buffer including, minimally, sugars, egg yolks, and antibiotics. It may be a buffer containing egg yolk (pg. 9, para. [0023]). An embodiment of the method includes the step of mixing recovered semen with a solution comprising 10% egg yolk. After cooling the mixture, a cryoprotectant comprising 4% LDL is added to the sperm diluent (pg. 13, para. [0039]).
That is, Oh et al. shows a semen extender composition comprising both egg yolk and low-density lipoprotein.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have modified the method of reducing agglutination and improving the health of sperm cells having an altered ratio of viable X and Y chromosome-bearing sperm cells by incorporating the use of egg yolk, as shown by Durack et al., would: 1) reduce sperm agglutination as a result of performing the method with the described collection additives [Claim 1], as shown by Gomes-Alves et al., with a reasonable expectation of success, because Gomes-Alves et al. shows compositions which include egg yolk for improving several characteristics of collected sperm, which are the compositions used in the method shown by Durack et al. (MPEP 2143 (I)(G)).
One of ordinary skill in the art would have been motivated to have included egg yolk in the extender compositions, because Gomez-Alves et al. also shows that other characteristics of sperm that are prepared for cryopreservation, such as motility, viability and high mitochondrial potential, can also be improved by using the egg yolk-containing extenders. One of ordinary skill in the art would have been motivated to modify the method disclosed by Durack by also incorporating a low-density lipoprotein additive [Claim 1], as shown by Moreno et al., with a reasonable expectation of success, because Moreno et al. teaches that LDL represents the cryoprotective fraction of egg yolk, can be extracted from egg yolk, and shows that LDL can improve the motility and viability characteristics of sperm when added to a sperm or semen extender.
Durack et al. teaches that an egg yolk or egg yolk extract can be added to the collection medium in order to improve sperm health. Therefore, it would have been obvious to have incorporated the LDL shown by Moreno et al. into the method for improving the health of sperm cells, as shown by Durack et al., with the reasonably predictable expectation that the health of sperm cells would have been improved (MPEP 2143 (I)(G)).
In addition, Oh et al. shows a method for cryopreserving sperm which incorporates the use of a composition comprising both egg yolk (10%) and LDL (4%) (MPEP 2143 (I)(G)).
One of ordinary skill in the art would have been motivated to have made those modifications, because Moreno et al. teaches that egg yolk is used as a cryoprotective agent in semen freezing extenders, and shows that LDL also has cryoprotective activity. Therefore, one of ordinary skill in the art of sperm collection medium production would have recognized the added or synergistic benefit of adding LDL to a collection medium comprising egg yolk, as shown by Oh et al., in order to augment the protective effect of said medium on sperm cells.
Regarding claim 1, pertaining to media containing a wt/vol concentration of about 0.1% egg yolk,
Durack et al. teaches that the sorted sperm are collected in a vessel that contains a collection fluid. The collection fluid may comprise a protein source (pg. 103, lines 28-33). If included, the protein source may be any protein source that does not interfere with the viability of the sperm cells and is compatible with the particular buffer or buffered solution being used. Examples of common protein sources include, minimally, egg yolk and egg yolk extract (pg. 104, lines 3-6). Durack et al. teaches that such proteins, if included in the collection fluid, may be used in a concentration of from about 1% (v/v) to about 30% (v/v), and more preferably about 10% (v/v) (pg. 104, lines 6-8).
Although Durack et al. does not show egg yolk concentrations as weight/volume percentages and does not show egg yolk concentrations at about 0.1% (per claim 1), it would have been obvious to one of ordinary skill in the art to have determined a percent concentration of egg yolk as a weight/volume unit rather than a vol/vol percent, as shown by Durack et al., barring a showing of criticality for the specific limitation (MPEP 2144 (III)). In addition, it would have been further obvious to have used routine optimization to have determined that the optimal percentage of egg yolk in the final collection fluid is about 0.1%, depending on, for example, the size of the sperm cell sample or to what extent said sample was diluted (MPEP 2144.05 (II)(III)).
Regarding claim 2, pertaining to i) removing at least a portion of one of either the viable X chromosome-bearing sperm cells or the viable Y chromosome-bearing sperm cells from the sperm cell sample,
Durack et al. shows that the collection system operates to intercept the droplets containing sperm in the sorted streams (pg. 102, lines 6-7). One (of two) intercepting devices for each cytometry unit can direct fluid into a common waste trough rather than a collection vessel for discarding sorted droplets that contain particles of little value (e.g., Y-chromosome bearing sperm cells for breeding dairy cows) (pg. 134, lines 10-13).
Regarding claim 3, pertaining to injecting the stained sperm cell sample into a flow of sheath fluid,
Durack et al. shows that the stained cells may be introduced into the sheath fluid at any time subsequent to staining (pg. 30, lines 27-28).
Further regarding claim 3, pertaining to exposing the stained sperm cell sample in the flow of sheath fluid to an electromagnetic radiation source that causes a detectable response in the DNA selective dye, detecting the response of the DNA selective dye to the electromagnetic radiation exposure, analyzing the detected response,
Durack et al. shows that the optics system is adapted for focusing a beam of electromagnetic radiation (e.g., a laser beam) on the fluid/flow stream (pg. 46, lines 7-8). The fluid/flow stream carries the (sperm) particles through a focused beam of electromagnetic radiation (e.g., UV laser light), and the electromagnetic radiation (e.g., fluorescent light) emitted by particles passing through the focused beam is analyzed (pg. 31, lines 21-23).
Further regarding claim 3, pertaining to classifying sperm cells in the sperm cell sample based on the analysis of the detected response,
Durack et al. shows that a period of time for dyeing the sperm cells is typically a period sufficient for the dye to bind to the DNA of the sperm cells such that X and Y chromosome-bearing sperm cells may be sorted based upon the differing and measurable fluorescence intensity between the two (pg. 29, lines 19-22).
Regarding claim 18, while a particular process described is in the context of sorting animal sperm (e.g., bovine sperm), it will be understood that the various aspects of this invention are more generally applicable to any type of sperm (equine, porcine, and others) (pg. 23, lines 18-21).
Regarding claim 19, the sperm are preferably collected or quickly transferred into an insulated container to avoid a rapid temperature change from physiological temperatures (typically about 35°C to about 39°C) (pg. 23, lines 33-35).
Regarding claim 20, at the time of the introduction of the stained cells to the sheath fluid, both the cells and the sheath fluid are at the same temperature; for example, at a "relatively low" temperature, such as for example at about 5°C to about 8°C; or at an "intermediate" temperature, such as for example at about 25°C to about 30°C (pg. 31, lines 1-4). Conventional cryopreservation methods involve sequentially adding a protein source (e.g., egg yolk), cooling the sperm to a temperature of about 4-5°C, adding a cryoprotectant (e.g., glycerol), maintaining the sperm and cryoprotectant at a steady temperature in the range of about 4-5°C for a period of time sufficient to allow the sperm cells to equilibrate with the cryoprotectant (pg. 115, lines 37-40 thru pg. 116, line 1).
Although not shown by Durack et al., it would have been obvious to have conducted the step of collecting at a temperature less than 22°C [Claim 20], with a reasonable expectation of success, because Durack et al. does show that other steps performed in the method of improving the health of sperm cells are conducted at lower temperatures (e.g., cooling the sperm to a temperature of about 4-5°C before adding a cryoprotectant, and preparing the sperm for flow cytometry at 25°C to about 30°C), so as to prepare the collected sperm cell population for subsequent cryopreservation. Therefore, it would have been obvious (and one of ordinary skill in the art would have been motivated) to have collected the at least one altered sperm cell population at a temperature of less than 22oC so as to minimize the ‘temperature shock’ that the sperm cell population would have to endure before being frozen/cryopreserved.
Regarding claim 1, pertaining to specific w/v percent concentration of LDL at about 2.5%,
Moreno et al. shows that eight (8) extenders were prepared with different concentrations of LDL (0.25%, 0.50%, 0.75%, 1%, 2%, 3%, 4% and 5%) (pg. 1068, Abstract).
Although Moreno et al. does not show that the percent concentrations of LDL are based on a wt/vol calculation, Table 1 shows that the various extender compositions were made with LDL in gram amounts and the other components (e.g., egg yolk) in milliliter amounts (pg. 1070, Table 1). Therefore, it would have been obvious to have considered the LDL percentages, shown by Moreno et al., to be w/v percentages (MPEP 2144 (I)).
Although Moreno et al. does not show 2.5% it would have been obvious to have tried an LDL concentration of 2.5%, because the range of LDL percentages, shown by Moreno et al., includes 2.5% (MPEP 2143 (I)(G)).
3. Claim 21 remains rejected under 35 U.S.C. §103 as being unpatentable over Durack et al. in view of Gomes-Alves et al., Moreno et al., and Oh et al., as applied to claims 1-4 and 18-20 above, and further in view of Wang et al. ((2018) Poultry Sci. 97: 2230-2238).
Durack et al. in view of Gomes-Alves et al., Moreno et al., and Oh et al., as applied to claims 1-10 and 12-20 above, do not show: 1) the low-density lipoprotein additive is extracted using polyethylene glycol [Claim 21].
Wang et al. teaches that low-density lipoprotein (LDL) from hen egg yolk has high nutritional value and plays an important role in the fields of biology, medicine, and materials (pg. 2230, Abstract [nexus to Moreno et al. - LDL extracted from egg yolk]).
Regarding claim 21, Wang et al. shows a highly efficient method for extracting LDL from egg yolk. Polyethylene glycol (PEG) was selected to obtain crude LDL. Data indicate that PEG can extract more crude LDL when compared to other compounds used for LDL extraction (e.g., ammonium sulfate precipitation (ASP)) (pg. 2230, Abstract).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have modified the method of reducing agglutination and improving the health of sperm cells having an altered ratio of viable X and Y chromosome-bearing sperm cells by incorporating the use of egg yolk and low density lipoprotein, as shown by Durack et al. in view of Gomes-Alves et al., Moreno et al., and Oh et al., as applied to claims 1-10 and 12-20 above, by: 1) performing the low density lipoprotein (LDL) extraction from egg yolk with polyethylene glycol (PEG) [Claim 21], as shown by Wang et al, with a reasonable expectation of success, because Wang et al. teaches a(n) LDL extraction process for the purpose of producing a(n) LDL additive from hen egg yolk, and Moreno et al. show an extracted LDL additive from egg yolk (MPEP 2143 (I)(G)).
One of ordinary skill in the art would have been motivated to have made that modification, because Wang et al. teaches that the LDL extraction method using PEG produced more LDL than the ammonium sulfate precipitation method (ASP). Therefore, one of ordinary skill in the art of LDL production would have been motivated to have used an extraction method that results in the highest yield of LDL (MPEP 2143 (I)(C,D)). In addition, Durack et al. teaches that suitable cryoprotectants as cryoextenders include ethylene glycol (pg. 115, lines 5-10). Therefore, one of ordinary skill in the art of developing a method of reducing agglutination and improving the health of sperm cells having an altered ratio of viable X and Y chromosome-bearing sperm cells by incorporating the use of egg yolk and low density lipoprotein would not be concerned if some of the PEG compound was carried over into the LDL extract, because PEG would have no detrimental effects on the sperm collection medium composition, since Durack et al. states that it can be included in such a composition.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention.
4. Claim 22 remains rejected under 35 U.S.C. §103 as being unpatentable over Durack et al. in view of Gomes-Alves et al., Moreno et al., and Oh et al., as applied to claims 1-4 and 18-20 above, and further in view of Banihani ((2017) Biomolec. 7(42): 1-10).
Durack et al. in view of Gomes-Alves et al., Moreno et al., and Oh et al., as applied to claims 1-10 and 12-20 above, do not show: 1) the collection media further comprises vitamin B or alpha-ketoglutarate [Claim 22].
Regarding claim 22, Banihani teaches that various studies have revealed the effects of vitamin B12, also named cobalamin, on semen quality and sperm physiology (pg. 1, Abstract [nexus to Durack et al.- method for improving sperm health]). The mainstream published work demonstrates the positive effects of vitamin B12 on semen quality: first, by increasing sperm count, and by enhancing sperm motility and reducing sperm DNA damage. The beneficial effects of vitamin B12 on semen quality may be due to increased functionality of reproductive organs, decreased homocysteine toxicity, reduced amounts of generated nitric oxide, decreased levels of oxidative damage to sperm, reduced amount of energy produced by spermatozoa, decreased inflammation-induced semen impairment, and control of nuclear factor-kB activation (pg. 1, Abstract). The positive effects of vitamin B12 on sperm parameters (e.g., count, motility, morphology, sperm DNA) have been investigated in various studies. Table 1 presents a summary of the human studies done on vitamin B12 and its derived compounds (pg. 2, para. 2.1.1 and Table 1).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have modified the method of reducing agglutination and improving the health of sperm cells having an altered ratio of viable X and Y chromosome-bearing sperm cells by incorporating the use of egg yolk and low density lipoprotein, as shown by Durack et al. in view of Gomes-Alves et al., Moreno et al., and Oh et al., as applied to claims 1-10 and 12-20 above, by: 1) adding vitamin B to the collection media [Claim 22], as taught by Banihani, with a reasonable expectation of success, because Banihani teaches that there is extensive literature showing the beneficial effects of vitamin B-12 on semen quality, including the improvement of specific characteristics of sperm (e.g., sperm count and motility), and Durack et al. shows a method and compositions for improving the health of sperm cells (MPEP 2143 (I)(G)).
Therefore, it would have been obvious to have incorporated the vitamin B shown by Banihani et al. into the method for improving the health of sperm cells, as shown by Durack et al. in view of Gomes-Alves et al., Moreno et al., and Oh et al., as applied to claims 1-10 and 12-20 above, with the reasonably predictable expectation that the health of sperm cells would have been improved (MPEP 2143 (I)(G)).
One of ordinary skill in the art would have been motivated to have included vitamin B in the extender compositions shown by Durack et al. , Gomes-Alves et al., and Moreno et al., because Banihani shows that other characteristics of sperm (in addition to those shown by Gomes-Alves et al.) can also be improved by adding vitamin B12 to a sperm collection medium (e.g., sperm count, morphology and sperm DNA) (MPEP 2143 (I)(C,D)).
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention.
Response to Arguments
5. Applicants argue that the claimed invention yields unexpected results, and thus, is nonobvious over any of the cited combinations. Amended claim 1 is drawn to a method of sex-sorting sperm cells comprising "collecting the at least one altered sperm cell population in a collection media comprising egg yolk at a wt./vol. concentration of about 0.1% and a low-density lipoprotein additive at a wt./vol. concentration of about 2.5%. As shown in Examples 4C to 4E in the specification-as-filed, collecting sex-sorted sperm cells in a collection media comprising 0.1% egg yolk and 2.5% LDL yielded significantly improved sperm cell viability and reduced numbers of dead cells compared to the use of media comprising egg yolk or LDL alone. Applicants argue these results are clearly unexpected, because Moreno, as acknowledged by Examiner at page 24 of the Office Action, expressly teaches that components of egg yolk "exert a noxious effect on spermatozoa" (see Moreno at page 1069), which is the basis for Moreno's experiments using LDL instead of egg yolk. Accordingly, based on Moreno's express teaching, one of ordinary skill in the art would reasonably expect that a media comprising egg yolk and LDL would yield reduced sperm viability compared to a media comprising LDL alone-the exact opposite of Applicant's findings.
Applicants argue that Oh and Bertuzzi do nothing to alter this conclusion. Oh and Bertuzzi are expressly drawn to medias for cryopreservation and cooling of sperm cells, respectively. Neither reference teaches or suggests the use of such medias in a method of flow cytometrically sex-sorting sperm cells, as presently claimed, much less the benefits obtained by collecting sex-sorted sperm cells in the claimed media. Bertuzzi at page 1 only mentions the use of egg yolk in the context of preserving cooled sperm ("it is known that the addition of EY to some extenders improved the preservation of cooled sperm"). Similarly, Oh at page 9 only mentions the use of egg yolk in order "to prevent damage that may occur due to sperm collision during cryopreservation." Accordingly, one of ordinary skill in the art seeking to flow cytometrically sex-sort sperm cells-a method that does not comprise, much less require, cooling or cryopreservation-would have absolutely no reason to add egg yolk to a media used to collect sex-sorted sperm cells, as presently claimed.
6. The arguments have been considered but are not persuasive. Regarding the claimed composition comprising w/v about 0.1% egg yolk (EY) and about 2.5% LDL additive, Example 4C shows the comparison of 4 compositions in catch media post sort of boar sperm:
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The specification concludes from these results:
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Although Applicant argues the sex-sorted sperm cells in a collection media comprising 0.1% egg yolk and 2.5% LDL yielded significantly improved sperm cell viability and reduced numbers of dead cells compared to the use of media comprising egg yolk or LDL alone, there was no comparison in Example 4C of the claimed composition to a control composition comprising LDL alone. Further, the “0.1” composition comprising 0.1% egg yolk + 2.5% LDL, similar to claim 1, had NO significant difference in motility compared to control media containing only 20% EY. The “0.1” composition comprising 0.1% egg yolk + 2.5% LDL, similar to claim 1, had NO significant difference in motility, viability and death with the “0.5” composition comprising 0.5% egg yolk + 2.5% LDL. The “0.5” composition comprising 0.5% egg yolk + 2.5% LDL, falling in the scope of claim 1, had NO significant difference in viability and death with the control media containing only 20% EY or the “1” media containing 1% EY + 2.5% LDL. Further, the “0.5” composition comprising 0.1% egg yolk + 2.5% LDL, falling in the scope of claim 1, had NO significant difference in agglutination rank with the “1” media containing 1% EY + 2.5% LDL.
Additionally, although Table 7 indicates statistical difference in viability between the “0.1” composition (81.82) and the control containing only 20% EY (76.52), Table 7 also indicates NO statistical significant difference in viability between the “0.5” composition (78.56) and both the “0.1” and control compositions. Example 4C does not provide any comparisons of the “0.1” composition in viability, motility, and agglutination to compositions comprising only LDL or a wider range of EY and LDL concentrations to definitively support Applicants conclusion that the specifically claimed concentrations of about 0.1% EY + 2.5% LDL yield unexpected results compared to what it taught and expected by the cited prior art. The cited prior art teaches the expected functions and benefits of adding egg yolk and LDL to a sperm composition and at various concentrations to improve viability, motility, and aggregation for the reasons of record.
Applicant argued that the cited references teach the addition of EY and LDL to collected sperm media in the context of improving sperm viability, motility and aggregation through cryopreservation processes, and not through sperm sorting and collecting processes as claimed. Claim 1 recites the sorted and collected sperm is placed into a media that comprises the w/v ~ 0.1% EY + ~2.5% LDL, however, claim 1 does not exclude the subsequent cryopreservation and thawing of the sperm for which the cited art recognizes benefit of the composition for. The cited references teach and render obvious the claimed staining and sorting method steps for the reasons of record, and provide motivation to place the sorted sperm into a media containing w/v ~ 0.1% EY + ~2.5% LDL for improved viability, motility, and aggregation rates, even if intended for cryopreservation.
Similar to Example 4C above, Example 4D in the specification fails to demonstrate evidence of unexpected properties for the claimed method and sperm composition above and beyond what is taught by the cited prior art. Applicant's Example 4D shows the comparison of four compositions that comprise w/v 2.5% LDL but lower EY levels at 0.05% and 0.01% that may be reasonably encompassed by the instantly claimed “about 0.1%” EY:
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The specification concludes:
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Results show that % motility was higher with the 20% egg yolk control ("-Control") compared to both the 0.01 and 0.05 combination treatments. The 2.5% LDL control ("+Control") was higher than the 0.05 combination treatment, but not the 0.01 combination treatment. With regard to the % Viability, the +Control performed better than the 0.05 combination treatment. It is noted that except for the % Motility test, the results of the other five tests were deemed statistically significant (see "P Value" with values of < 0.05), despite the small differences in some of the numerical results (e.g., less than a tenth of a percent). Test results are attributed a "PSE" value (pooled standard error).
That is, either of the combination LDL + EY treatments did not consistently perform better than either of the controls. Despite the purported statistical significance, it is not clear that the medium comprising LDL and egg yolk exhibits (unexpectedly or surprisingly) improved results.
MPEP 716.02(d) states:
The nonobviousness of a broader claimed range can be supported by evidence based on unexpected results from testing a narrower range if one of ordinary skill in the art would be able to determine a trend in the exemplified data which would allow the artisan to reasonably extend the probative value thereof. In re Kollman, 595 F.2d 48, 201 USPQ 193 (CCPA 1979) (Claims directed to mixtures of an herbicide known as "FENAC" with a diphenyl ether herbicide in certain relative proportions were rejected as prima facie obvious. Applicant presented evidence alleging unexpected results testing three species of diphenyl ether herbicides over limited relative proportion ranges. The court held that the limited number of species exemplified did not provide an adequate basis for concluding that similar results would be obtained for the other diphenyl ether herbicides within the scope of the generic claims. Claims 6-8 recited a FENAC:diphenyl ether ratio of 1:1 to 4:1 for the three specific ethers tested. For two of the claimed ethers, unexpected results were demonstrated over a ratio of 16:1 to 2:1, and the effectiveness increased as the ratio approached the untested region of the claimed range. The court held these tests were commensurate in scope with the claims and supported the nonobviousness thereof. However, for a third ether, data was only provided over the range of 1:1 to 2:1 where the effectiveness decreased to the "expected level" as it approached the untested region. This evidence was not sufficient to overcome the obviousness rejection.); In re Lindner, 457 F.2d 506, 509, 173 USPQ 356, 359 (CCPA 1972) (Evidence of nonobviousness consisted of comparing a single composition within the broad scope of the claims with the prior art. The court did not find the evidence sufficient to rebut the prima facie case of obviousness because there was "no adequate basis for reasonably concluding that the great number and variety of compositions included in the claims would behave in the same manner as the tested composition.").
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In the instant case, in pointing to Example 4 of the specification, Applicants have not persuasively demonstrated their asserted unexpected results occur only for the instantly claimed range of egg yolk and LDL, and would not occur across a broader range as demonstrated by the cited prior art.
Contrary to arguments, the cited art does not teach away from adding egg yolk to the sperm media, but rather, the cited art teaches and exemplifies that egg yolk is a common, established protective semen extender, for the reasons stated in the rejection. Egg yolk (which contains low-density lipoprotein) has been used as a well-known semen extender ingredient, because it protects the sperm from thermal shock during freezing (Moreno et al. (cited above in the 103 rejection), pg. 1069, column 1, para. 1). On the other hand, it is acknowledged that despite the protective effect of egg yolk, a few of its components exert a noxious effect on spermatozoa (Moreno et al., pg. 1069, column 1, para. 1). Moreno et al. shows experiments in which the low-density lipoprotein (LDL) purified from egg yolk was added to semen extenders to determine the appropriate concentration of LDL to obtain the best post-thaw motility results, because it has been shown that the use of LDL (purified from egg yolk) in semen extenders (instead of whole egg yolk) improved the post-thaw motility rate of spermatozoa (in bull) (Moreno et al., see above). Further, Oh teaches mixing recovered semen with a solution comprising egg yolk and adding LDL for cryopreservation. Therefore, the cited prior art establish the known, predictable function of egg yolk and LDL (derived from egg yolk) as a semen extender and their benefits for semen compositions subsequently cryopreserved.
Therefore, it would have been obvious to one of ordinary skill in the art of producing a semen extender composition for use in a method of reducing agglutination and improving the health of sperm cells (and one would have been motivated) to have combined some low level of egg yolk (e.g., w/v about 0.1%) and a desired level of LDL into one composition, as shown by Oh et al. (cited in the 103 rejection above), so as to reap the benefits of both egg yolk (i.e., protection of sperm from thermal shock during freezing) and LDL (i.e., improvement in post-thaw motility rate).
7. All other rejections recited in the Office Action mailed November 17, 2025 are hereby withdrawn in view of amendments canceling claims. The rejection of claim 11 under 35 U.S.C. §103 as being unpatentable over Durack et al. in view of Gomes-Alves et al., Moreno et al., and Oh et al., and further in view of Ulrichs et al. ((2010) Arch. Geflügelk. 74(4): 279-284) is withdrawn in view of cancellation of claim 11.
8. Conclusion: No claim is allowed.
Conclusion
9. THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
10. Any inquiry concerning this communication or earlier communications from the examiner should be directed to LAURA B GODDARD whose telephone number is (571)272-8788. The examiner can normally be reached Mon-Fri, 7am-3:30pm.
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/Laura B Goddard/Primary Examiner, Art Unit 1642
