US20200267969A1 - Use of tree sap to preserve sperm cell lines - Google Patents

Abstract

A method of cryogenically preserving sperm comprising (a) combining sperm to be cryogenically preserved and a composition that comprises (1) a cryoprotectant, comprising one or more tree saps; and (2) an extender medium to produce a sperm/medium combination; and (b) subjecting the combination to conditions that result in cryopreservation of sperm, thereby producing a cryopreserved combination that comprises cryopreserved sperm is disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
• This application is a continuation of U.S. patent application Ser. No. 15/569,164 filed on Oct. 25, 2017, which represents the national stage entry of PCT International Application No. PCT/US2016/029351 filed on Apr. 26, 2016 and claims priority to U.S. Patent Application Ser. No. 62/153,197, filed Apr. 25, 2015, the contents of all of which are incorporated by reference as if set forth in their entirety herein.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
• N/A
BACKGROUND
• In general, the present invention involves the use of tree sap to cryogenically preserve avian and mammalian sperm cells, preferably for use in the poultry industry, birds of prey preservation, and preservation of endangered or threatened avian species. The present invention may also be used in the cattle industry, pig industry, equine industry, and in mammalian veterinary medicine.
• Avian spermatozoa have a shape that makes the spermatozoa hard to freeze. The spermatozoa are long and thin and are shaped like a whip. This makes the cells very subject to cryogenic injury because they have a large surface area that can be damaged easily upon freezing or processing. Mammalian sperm will also benefit from the present invention because even though these cells are easier to freeze, they are still subject to damage from the cryogenic processes. (Reference; Avian Semen Cryopreservation: What Are the Biological Challenges? J. A. Long, 2006 Biotechnology and Germplasm Laboratory, Animal and Natural Resources Institute, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, Md. 20705, 2006 Poultry Science Association, Inc. Accepted Sep. 10, 2005)
• Currently, avian spermatozoa are frozen using several techniques. One technique uses the addition of a cryoprotectant to a fluid media that suspends and supports the cells. The first step in the procedure is to collect the semen and then add a liquid extender. A semen extender is a liquid diluent which is added to semen to preserve its fertilizing ability. The extender allows the semen to be freighted to the female, rather than requiring the male and female to be near to each other. Special freezing extender also allows cryogenic preservation of sperm (“frozen semen”), which may be transported for use, or used on-site at a later date.
• This extender/cell mixture is then placed in a refrigerator to chill the mixture down to a desired temperature that allows the cells to line up the lipid components in their outer cell membrane prior to freezing. This is a form of “cold acclimation” and helps to allow the cells to survive the cryogenic process. The method also reduces the temperature gradient drop that the cells have to go through before they reach the freezing point and reduces the cell damage when being frozen.
• Once the cells are chilled/acclimated, the cryoprotectant is added to the extender/cell mix, the mixture is packaged quickly and either flash frozen by quick immersion in the liquid nitrogen, pelletized and flash frozen and then packaged into cryo-vials, or suspended above the liquid nitrogen in the vapors to freeze more slowly before it is immersed in the liquid nitrogen. Both fast and slow freezing can be done based on species requirements. Different cryoprotectants that are added to the mix commonly include DMSO (Dimethyl sulfoxide), MA (Methyl-Acetamide), and DMA (Dimethyl Acetamide). These chemicals act as intracellular cryoprotectants while the non-cell wall-permeable chemicals act as extracellular cryoprotectants. These are also known to damage the cell wall during cryopreservation and this impairs fertility.
• A better and more effective way of preserving avian and mammalian semen is needed in the art.
SUMMARY OF THE INVENTION
• In one embodiment, the present invention is a method of cryogenically preserving sperm comprising (a). combining sperm to be cryogenically preserved and a composition that comprises (1) a cryoprotectant, comprising one or more tree saps; and (2) an extender medium to produce a sperm/medium combination and (b). subjecting the combination to conditions that result in cryopreservation of sperm, thereby producing a cryopreserved combination that comprises cryopreserved sperm. In one version of the invention the sperm is avian sperm. In one version the sperm is derived from the Northern goshawk (Accipiter gentilis).
• In another version of the invention the sperm is derived from a mammalian non-human species, preferably selected from the group consisting of cattle, pigs and equines.
• In one version of the invention the sap is either maple tree sap or birch tree, preferably both first run saps.
• In one version, the present invention is the cryopreserved combination resulting from the method described above.
• In another version, the present invention is a method of fertilizing an egg cell comprising the step of introducing the combination described above to an unfertilized egg cell, wherein the egg becomes fertilized.
DESCRIPTION OF THE INVENTION
• In general, the present invention is a method and medium useful for the cryogenic preservation of sperm using tree saps. In another embodiment, the present invention is a composition comprising the mixture of the preservation medium and the sperm, using tree saps.
• Although the present invention is useful for all animal sperm, the invention is most preferably used with avian sperm because of the special physiologic needs of the avian samples. Preferred avian species include birds of prey (such as Falconiforms and Strigiformes) and commercial species such as turkeys, chickens (Galliformes) and ducks (Anseriformes.) Other preferred avian species include but are not limited to Passeriformes and Psittacifomes.
• In another version of the invention, one may wish to preserve the sperm of other mammalian species, including cattle (Family—Bovidae), pigs (Family—Suidae), horses (Family-Equidae) and veterinary medicine applications, including canine (Canidae) and feline (Felidae) species.
• In certain embodiments, the method of cryogenically preserving sperm comprises: (a) combining sperm to be cryogenically preserved with a medium comprising (1) a cryoprotectant, such as one or more tree saps or its extracts; and (2) an extender designed to support cell life, wherein the combination produces a sperm/medium combination (cryoprotective medium/sperm combination); and (b) subjecting the combination to conditions that result in cryopreservation of sperm, thereby producing a cryopreserved combination that comprises cryopreserved sperm.
• The typical sperm extender typically contains chemicals to both stabilize and protect cell membranes. The Examples below use Beltsville Turkey Extender (BTE) recipe with the exception of removing fructose as one of the ingredients. The fructose was replaced with sucrose and constitutes a separate extender recipe, also a preferred embodiment of the present invention. It was found that goshawk semen did not do well with fructose as its energy source when being cryogenically preserved. This observation is true for other animal cell lines.
• The preferred extender recipe for goshawk semen, and other typical avian semen samples, consists of;

• 	Potassium Diphosphate 3H2O	12.7	grams
  	Sodium glutamate	8.675	grams
  	Sucrose to replace Fructose (Anhydrous)	5.000	grams
  	Sodium Acetate 3 H2O	4.255	grams
  	TES	1.95	grams
  	N-tris Hydroxymethyl Methyl-2Amino-
  	ethane Sulfonic Acid
  	Potassium citrate	.64	grams
  	Potassium Monophosphate	.65	grams
  	Magnesium Chloride	.338	grams

  	Purified water	1022 ml is added to
  		the dry ingredients.

• This constitutes a full recipe of the preferred extender for goshawk semen for cryopreservation. The sucrose is often left out of this recipe and supplied just through the addition of tree saps that naturally have sucrose in them. Other base recipes may be preferred for other cell lines in other species to meet those species specific requirements. The sugar supplied for the recipe may come from the sap as in the Examples list in the Excel Spreadsheet.
• The present invention involves the use of tree sap as a cryoprotectant. Tree sap is a fluid transported in xylem cells (tracheids or vessel elements) or phloem sieve tube elements of a plant. Two kinds of sap are defined as either Xylem sap or Phloem sap. We include both kinds of sap in our definition.
• Tree sap is produced at a time of the year when the trees are going through cold stress and freezing in the temperature ranges that are most harmful to the cells that we are trying to freeze. The trees survive temperatures from freezing to minus 60° F. The trees also survive the daily shift in temperatures that the tree can survive both well above freezing to well below it. The tree sap contains properties that allow it to support cell life even when frozen and when going through rigorous freeze thaw cycles and daily temperature extremes. It contains various sugars, antifreeze proteins, carbohydrates, minerals, phenolic compounds, and other compounds that provide cryoprotective properties. Some of these compounds have yet to be described.
• The tree species that are most useful in this invention includes the cold-hardy maple tree species, birch tree species, poplar tree species, aspen tree species, and other trees that can be tapped or where chemicals or fluids can be extracted from them. Tree species from the higher latitude deciduous forests are included even if not listed directly herein.
• A common factor in these trees is the amount of sugar in the sap. Sugars have cryoprotective properties. Some avian extender recipes often call for 0.5% of either sucrose or fructose. Most tree species meet or exceed this percentage sugar requirement. Maple tree range anywhere from approximately 0.5% to 4.0% sucrose. Another common factor that these trees have is that they have non-sugar cryoprotectant chemicals in their sap. These chemical may provide stronger cryoprotective properties than that the simple sugars that are easily measured.
• There are over 128 species of maple trees worldwide. The sugar maple (Acer saccharum) and black maple (Acer nigrum) produce the most sugar in their saps. The red maple (Acer rubrum) and the silver maple (Acer saccharinum) produce less sugar but are in the latitudes where they will likely contain similar cryoprotective properties in their sap. These later two species are one preferred version of the present invention due to the lower sugar content and potentially higher cryoprotective properties in the sap that are not from sugars.
• Birch tree (Family-Betulaceae, Genus-Betula), poplar tree (Family Salicaceae, Genus-Populus), and aspen tree (Family-Salicaceae, Genus-Populus) species come from higher latitudes in the United States and Canada and have lower sugar content in their sap than the maple (Acer species) tree species do. The non-sugar cryoprotective chemicals in their sap will likely be higher as these species survive in a more extreme environment with temperature ranges fluxuating widely below freezing, and the trees have a lower content of the sugars that are known to be cryoprotective in their sap.
• Twenty three species of trees that can be tapped in the United States and are useful in the present invention include but are not limited to Sugar Maple (Acer saccharum), Black Maple (Acer nigrum), Red Maple (Acer rubrum), Silver Maple (Acer saccharinum), Norway Maple (Acer platanoides), Boxelder (Acer negundo), Bigleaf Maple (Acer macrophyllum), Canyon Maple or Big Tooth Maple (Acer grandidentatum), Rocky Mountain Maple (Acer glabrum), Gorosoe (Acer mono), Butternut or White Walnut (Juglans cinerea), Black Walnut (Juglans nigra), Heartnut (Juglans ailantifolia), English walnut (Juglans regia), Paper Birch (Betula papyrifera), Yellow Birch (Betula alleghaniensis), Black Birch (Betula lenta), River Birch (Betula nigra), Gray Birch (Betula populifolia), European White Birch (Betula pendula), Sycamore (Platanus occidentalis), Acer ginnala, and Ironwood or hophornbeam (Ostrya virginiana).
• Preferably, one would begin with extender recipes designed for the preservation or storage of animal sperm. Typically, the initial amount of sap added to the modified extender recipe will make the initial solution physiologically close to the osmolality of the raw semen and still provide for cryo-protection of the cells. The initial osmolality range needed is determined by the measurement of the osmolality of the raw semen.
• Goshawk semen has an osmolality of about 341 mili-osmoles. Later additions of extender/sap combinations increase the osmolality of the mixture to dehydrate the cells immediately prior to freezing. Dehydrating the cells just prior to freezing them, increases survival.
• An ideal osmolality level is determined by the end results of the survival of the cells in question and the ability of the stored sample to create fertile female gametocytes. It is known that different species of birds have sperm cells that tolerate different osmolality extremes. Some avian spermatozoa survive very high osmolality and others do not. [See Species Variation in Osmotic, Cryoprotectant, and Cooling Rate Tolerance in Poultry, Eagle, and Peregrine Falcon Spermatozoa; Juan M. Blanco, George Gee, David E. Wildt, and Ann M. Donoghue; Biology of Reproduction Oct. 1, 2000 vol. 63 no. 4 1164-1171] The use of the sap allows both the removal of other toxic cryoprotectants from the mix and/or reduces the amount of toxic cryoprotectants used. Yet, one may still wish to add additional cryoprotectants to the mix.
• A typical sperm/sap-extender combination of the present invention is as follows: The final volume of the sperm/sap-extender combination should be no more than 1:3 dilutions making the semen a quarter of the final volume. Semen dilutions higher than this can impair fertility because of simple dilution.
• In a preferred version of the invention, sap comprises at least 30% of the final sperm/extender combination. In another version of the invention, sap comprises 10%-80% of the final sperm/extender combination, preferably about 50%.
• Over dilution reduces sperm fertility of the sample. The amount of sap needed to provide cryogenic protection to the mixture varies considerably because of tree species variation in cryoprotectant types and properties.
• I have been modifying the extender recipe enough to allow sap to be blended into the mix so that this mix then supports the cells when they are frozen in liquid nitrogen (LN₂) with or without the use of an additional cryoprotectant. A typical example of extender includes the dry ingredients of the Beltsville Turkey Extender without the fructose (BTE minus fructose) as a base recipe to work with.
• The sap from the Maple tree and the Birch tree were then added to the BTE minus fructose and used at different ratios, to preserve the sperm cells. A recipe that preserved the cells in LN₂ well included 1 part raw semen, 1 part BTE minus fructose with 0.5% sucrose added back in, 2 parts BTE minus fructose with sap added, to supply its liquid volume. The sperm and the BTE minus fructose with 0.5% sucrose added back in; were mixed in a 0.5 ml Eppendorf vial in the fridge. A matching volume of BTE minus fructose with sap as its liquid diluent was also placed in the fridge but in a separate tube. Both tubes were allowed to equilibrate to an equal temperature for 10 minutes before they were then mixed, packaged, and then flash frozen. The work was done in the fridge at 42° F. so there were no temperature fluxuations to stress the semen. This form of cold “acclimation” allowed the lipid component of the cell wall to line up prior to freezing to help prevent damage to the cell structure.
• Therefore, a preferred version of the present invention comprises a composition, wherein 1 part of the volume is raw semen; 1 part of the volume is extender, such as BTE no fructose plus 0.5% sucrose added back in; and 2 parts of the total volume was BTE no fructose with sap of either the maple or birch tree. A minimum of 50% sap by volume should preferably be used in this mix. Samples with 50% sap by volume had far better survival on thaw than samples with less than this percentage.
• The packaging consisted of the semen being placed in a 75 ul Mylar coated capillary tube with one end being caulked. Its opposite end was left open. This capillary tube was then placed inside a standard plastic poultry straw and the end opposite of the cotton was crimped shut. The poultry straw was then placed inside a plastic soda straw that had holes cut in the side of it. These holes allowed the LN₂ to enter and surround the poultry straw quickly as it was immersed. The holes in the soda straw also allowed the package to drain and breathe as it was thawed so that it did not explode.
• Cryopreservation can be carried out at any time after production of the medium/sperm combination as long as the storage does not significantly adversely affect the viability of the sperm. For example, cryopreservation can often be carried out as long as 180 minutes after the sperm/medium combination is produced with no loss of fertility. Samples should be chilling to extend the shelf life before freezing. A typical temperature for storing avian semen at is 5° C. Chilling the semen helps to line up the lipid component in the cell wall prior to freezing. This increases cell survival.
• Preservation is typically carried out at a temperature minus −198° C. In specific embodiments, cryopreservation is carried out at a temperature between from about minus −80° C. to about minus −198° C. In one preferred embodiment, the cryo-protection takes place in a liquid nitrogen bath/canister and the vials are stored at a −198° C. Long term storage can be achieved by placing the storage vials or straws in a liquid nitrogen canister. One would then wish to use the preserved sperm to fertilize a female gametocyte, female germ cell or ovum.
• Before the sperm is used for artificial insemination or incubated with a female gamete, the sperm is typically thawed and may also be washed. Sperm samples are often thawed in cold water or warm water baths with the temperature requirements being determined both by the species cell requirements or the cryoprotectant type used in the mix. Avian spermatozoa are typically thawed in ice water baths or cool water baths, and bovine spermatozoa are typically thawed in warm water baths that are body temperature. Insemination is performed immediately after thaw. Sperm are sometimes concentrated into pellets with the contents of different straws being combined, centrifuged down, to form a pellet of semen.
• In all embodiments described herein, the resulting cryopreserved sperm can be stored indefinitely.
• The fertilization capacity or ability of sperm can be assessed using methods known to those of skill in the art, such as in vitro methods, including assessing the ability to fertilize the oocytes/female gamete with which they are combined/incubated (their ability to form-cell embryos, for example) and/or in vivo methods, including assessing the production of offspring by females into whom the fertilized oocytes/female gamete are implanted (mammals). Fertilization capacity or ability can be assessed using available methods, such as a functional assay, including, but not limited to, a motility assay, a viability assay, a hemizona assay (binding of the sperm to the zona pellucida) or sperm penetration into zona-free mammalian or avian oocytes.
• The commercialization of cryogenically freezing avian semen has eluded scientists for decades. The freezing process has not been successful enough. Current papers cite approximately 35 to 40% semen motility after thaw. See; Comparative cryopreservation of avian spermatozoa; Benefits of non-permeating osmoprotectants and ATP on turkey and crane sperm cryosurvival. By Juan M. Blanco, Julie Long, George Gee, David E. Wildt, Ann M. Donoghue, Received 24 May 2010 Accepted 10 Dec. 2010. Elsevier B. V.
• The present invention, comprising the improvement of using sap as the sole cryoprotectant, often showed greater than 50% survival based on Live/Dead stains done after the thaw of samples. Some examples showed up to 73% survival on thaw with no additional cryoprotectant being used. Once this successful idea is combined with the other currently successful ideas of science, the survival of the semen will likely be high enough to make avian semen cryopreservation a commercially viable venture.
• Other animal species will likely benefit from this invention as well. There are numerous articles on scientists trying to freeze the semen of other animal species with limited success. The use of tree sap harvested at winter's first thaw, and used in cryopreservation of cell lines is an exciting and now documented success. The success of this process must also be evaluated based on the improved fertility and hatchability of eggs produced from females inseminated with frozen semen.
EXAMPLES
• In general, the present invention involves the use of tree sap to cryogenically preserve avian sperm lines, preferably for use in the poultry industry, birds of prey preservation, preservation of endangered or threatened avian species, and other avian species. It will also be useful in pigs (Family-Suidae), cattle (Family-Bovidae), horses (Family-Equidae), dogs (Family-Canidae), and cats (Family-Felidae).
• Table 1 contains the results of many experimental trials. In general, I obtained maple tree sap from the native trees in southeastern Wisconsin. The osmolality of Maple tree #3 is 100 mili-osmoles.
• The raw dry ingredients for the preferred medium were as follows: Beltsville Turkey Extender recipe, minus the fructose; had maple tree or birch tree sap added for a final volume of 100 ml. (I added 90 ml of sap to make the final volume of the dry and wet ingredients total 100 ml.)
• I began with a set of dry ingredients that was for 1/10^th of the standard recipe listed above. I added 90 ml of maple tree sap to one jar and 90 ml of birch tree sap to another jar to make a final volume of 100 ml in each jar. No other cryoprotectant was added into the mix. The fructose had been removed so the energy source for the semen came from the sucrose that was already in the sap. The cells that I am trying to preserve do not appear to metabolize fructose well and need the sucrose in the recipe to survive the freezing.
• The sap was used full strength in the stock jars, but it was used in different ratios when it was added to raw semen. Sometimes a 1:1:2 dilution was used (1 part semen: 1 part BTE no fructose, plus 1/% sucrose: 2 parts BTE plus Sap); sometimes a 1:1:1 dilution was used. Sometimes a 1:1:1 dilution was used where the final mix was 33% Semen and 66% sap with sap being added into the both the base mixture and the final mixture before freezing. In all cases no other cryoprotectant was added to the sample and only the sap was used to preserve the cells in the liquid nitrogen. The cells survived in large percentages even when no additional (penetrating or non-penetrating) cryoprotectant was added to the mix.
• Experiments were also done using the sap from Alaskan birch tree. Again, the raw dry ingredients for the Beltsville turkey extender, minus the fructose ( 1/10^th volume of the standard recipe) had birch tree sap added for a final volume of 100 ml. [The recipe for a standard liter volume of BTE is listed above.] No other cryoprotectant was added into the mix.
• The maple tree sap had been stored in 100 ml plastic bottles, with about 16 bottles per cardboard box, with the top left open. The top of the bottle had the minerals and other chemicals forced out of it leaving the ice crystals at the top. The center of the bottle had not frozen, and it remained in an almost glass like state without freezing completely after 24 hours. This type of freezing is critical to success when doing cryogenic freezing. This prevents ice crystal formation that damages the cells. [Reference; Investigation of Chemical and Physical Properties of Southwestern Wisconsin Maple Syrup; By Hiroyuki Takano, A Thesis Submitted in Partial Fulfillment of the Requirements for the Master of Science Degree with a major in Food and Nutritional Sciences. Martin G. Ondrus, Thesis Adviser; the Graduate School University of Wisconsin-Stout, December 2005]
• The sap of the birch tree was obtained from Alaska through a syrup company called Alaska Wild Harvest LLC, dba Kahiltna Birchworks, PO Box 2267, Palmer, Ak. 99645. I obtained both the first run and second run saps for experimentation. This sap contains three times less sugar on average, than Maple tree sap. This tree comes from higher latitudes that are subject to more severe temperatures and temperature swings than the forest in Wisconsin are. The birch tree sap froze very slowly in the chest freezer and in a similar manner to that of the maple tree sap listed in the previous paragraph.
• The first semen sample that I froze was from a male Northern goshawk (Accipiter gentilis) using an extender recipe that was modified to include maple tree sap. This recipe consisted of all of the dry ingredients of the Beltsville Turkey Extender in the usual percentages, without the fructose. 90 ml of maple tree sap was added for a final volume of 100 ml. (This is 1/10^th of a standard recipe for BTE) One hundred percent of the liquid added into the recipe was maple tree sap. The sucrose content of the maple tree sap is reported in the literature to be between 2%-2.6%. The exact sucrose level of this sap was not measured but estimated to be about 2% because this was a first run sap. The needed minimum sugar level for the Beltsville Turkey Extender is 0.5%. So this recipe ended up having more sugar in it (than the commercial extender) because the sap had 4-5 times the needed sugar level, naturally in its sap.
• This first recipe contained 4-5 times the needed sugar, making it hyperosmolar so that the sperm would gradually lose motility at room temperature. However, a frozen semen sample (Sample #69, Table 1) from the Northern goshawk (Accipiter gentilis) was immediately flash frozen upon mixing with the extender-sap combination 1:2 (1 part semen and 2 parts Extender/Sap combination) and 58% of the cells survived the freezing and thawing process based on a live/dead stain (Eosin/Nigrosin) and visual observations. This sample was thawed in a cool water bath at approximately 55° F. after being in the liquid nitrogen can for over a day. These cells then went on to lose motility at nearly at exactly the same rate as a sample that had been mixed and held at room temperature due to the chemical makeup of the sample. However, the cells survived the freezing process essentially unchanged. The motility and linear movement of the cells was left nearly intact, being unaltered by the freezing process. This was my first documented success and it exceeded my expectations. Cell survival post freezing showed great success.
• There was no other cryoprotectant put into the sample. This recipe is clearly hyperosmolar (and detrimental to the cells) because it contained at least 2% sucrose and the sperm only needed 0.5% sucrose.
• I found 58% survival upon thaw based on a live/dead Eosin/Nigrosin stain on this first sample. A hundred cells were counted using a standard lab cell counter and this simple percentage established. This exceeded literature references of 25% with standard cryoprotectants such as DMA and MA. A survival rate of above 25%, preferable above 40% or 50%, indicates a successful experiment.
• A second sample of 22 ul semen (sample #81, Table 1) was then frozen. 22 ul Beltsville Turkey Extender, no fructose, plus 0.5% sucrose was added to the semen in a 0.5 ml Eppendorf tube and placed in the fridge. 44 ul of BTE with maple tree sap; was placed in its separate tube, also in the fridge at 42° F. The two liquids were combined after acclimating in the fridge for 10 minutes. The total volume was 88 ul. The sample was packaged in 2-75 ul Mylar coated capillary tubes, placed in poultry straws, this was then placed in a ventilated soda straws, and flash frozen. Both straws were thaw in a 55° F. water bath. Two straws were produced from one semen sample. Sample 1 had 25-30% live forwardly motile sperm with normal speed of travel and a live/dead stain of 50 live/50 dead. The second straw had 55% forwardly motile with normal motility and a live/dead stain of 57 live/43 dead. The semen survival increased when the percentage of the Maple sap was lowered to 50%.
• Additional samples of semen from this male goshawk were frozen. The results are listed in Table 1, a table disclosing semen samples where either maple sap or birch sap were used exclusively for cryopreservation. The semen of the Northern goshawk (Accipiter gentilis) was used in all experiments.
• I list samples in Table 1 that are successful and those that are not in the column marked “Is this sample workable?” Samples were listed as Yes, No, and Maybe. There is a column that lists the success or lack of success; so it is easy to review the table quickly by looking down this single column. I had success freezing samples in LN₂ as soon as I started to add the natural Maple tree sap into the formula. Semen cells survived cryogenic freezing when only tree sap was used as the cryoprotectant, even when there was no other chemical cryoprotectant used. My samples survived the trauma of freezing almost as if they had never been frozen; continuing to swim at a normal speed in a straight direction. The cells eventually lost motility due to problems associated with the solution that they were put in.
• It is of particular note that some of the samples survived with even higher survival percentages and motility without the addition of other cryoprotectants, where only the sap was used. Sample 84 survived the best and is nearing the noted highest percentage of survival and motility known to scientists that work in this field of cryopreservation at 73 L/27 D % live/dead stain and a second sample with a 64 L/36 D % live/dead stain. Samples 67, 69, 70, 71, 72, 80, 81, 84, 93, 97, 118, 120, 126, and 128 show very encouraging results with progressive forward motility and the live dead stain percentages listed in the chart above. Other samples also showing this trend are also listed in the table below.

• Percentage Survival
  on Live/Dead Stain	Sample numbers in Table 1
  0-4% Survival	106, 113, 116, 117
  5-9% Survival	82, 100, 101, 105, 109, 110, 115, 116
  10-14% Survival	82, 88, 95, 105, 107, 110, 111, 111,
  	113, 114
  15-19% Survival	87, 88, 93, 100, 107, 108, 109, 115
  20-24% Survival	82, 91, 100, 102, 103, 106, 108
  25-29% Survival	99, 99, 114, 116, 117
  30-34% Survival	72, 77, 83, 89, 90, 90, 102, 109, 109,
  	119, 119
  35-39% Survival	44, 77, 77, 85, 91, 92, 99, 111
  40-44% Survival	68, 85, 80
  45-49% Survival	72, 83, 118
  50-54% Survival	81, 97, 103, 105, 107, 110, 112
  55-59% Survival	69, 80, 81, 93, 93, 128
  60-64% Survival	84, 120
  65-69% Survival	72, 76
  70-74% Survival	84

• Some of the samples showed quiescense features and according to a live/dead stain, survived the freezing but were not motile. Live cells do not take up Live/Dead stain and show up as white on microscope slides, even when no longer motile. These samples are likely not dead and can be“resurrected” and made motile with known techniques. Many of the samples had cells with near normal gross cellular features post freezing and did not appear to be distorted or damaged from the freezing process; on the live/dead stains. These stains have been retained for future reference.
• The sap is the key ingredient for cryopreservation because it is non-toxic, has no contagious agents to transmit to the sperm, is plentiful, has key cryo-preservative properties, is in a liquid state naturally, can be collected without bacterial contamination, and it is not viscous (thick) so it does not impair spermatic motility through the fluid medium. It is a natural product that is very unlikely to contain adulterant chemicals.
• I envision typical optimizations of the present invention. First, the optimized-liquid base that supports the cell lines needing to be preserved will need to be developed and then modified to allow the addition of the sap to the mix in various percentages, so that the sap does not add chemicals in concentrations that would then kill the cells, but would still allow for cryo-protection. (For example, the osmolality of maple sap that I obtained was 100 mili-osmoles.) This osmolality appeared to be too high when it was added directly to Beltsville Turkey Recipe dry ingredients that did not have the fructose added into the recipe. The cells survived the freezing in great shape, but lost motility possibly due to the hyperosmolality of the approximately 2% sucrose in the maple sap.)
• Second, the sap ingredient may be optimized just by choosing different species of trees to use. The sugar content in the saps varies with the tree species and so do the other chemicals that act as natural cryoprotectants that are not sugars. Syrup producers use maple trees that produce the most sugar and some syrup producers use birch trees for this process. They know that where maple tree sap is boiled down, between 20-50 units per one unit of syrup is required. When birch tree sap is boiled down, 150 units per 1 unit of syrup is required. Syrup producers do not tend to use maple tree species that produce low sugar content in their sap. Yet, these trees also survive the rigorous temperatures and temperature extremes and must be adapted well to survive without sugar as a main cryoprotectant, implying that other chemicals in the sap that are not sugars, are acting in this manner.
• Additionally, one might wish to use a combination of saps. Combinations of the maple and birch sap recipes were used in experimentation listed in Table 1. High success rates were achieved with this combination.
• Additionally, sap taken at different times during the tapping process may yield some beneficial results. Later run saps are lower in the sugars seen in the earlier run, while the trees are still going through and surviving extreme low temperature stresses. The osmolality of the saps taken at different times may be of benefit.
• Additionally, extracts of the saps may yield benefits through the discovery of newly discovered antifreeze proteins or compounds that would be of use with this process. [Reference; When plant cells can survive ultra-low temperatures; Pawl M. Pukacki, Physiology of Abiotic Stress Laboratory, Institute of Dendrology, Polish Academy of Sciences, Kornik, Poland].
• By “sap,” we mean to include any concentration or dilution of tree sap. For example, the cryoprotectants in the sap can be concentrated. One preferable way of concentrating the sap is via reverse osmosis. This is a mechanism whereby the water is removed from the sap and the chemicals are concentrated on one side of a semipermeable membrane without having to apply heat to the sap that would likely destroy the chemicals that are cryoprotective.
• Suitable Extender Recipes for 2015 and Semen Survival Study.
• Number 1 recipe; Beltsville Turkey Extender, No fructose plus 90 ml Maple Tree sap, from first run tapping, QS to 100 ml. Dated on bottle Mar. 11, 2015, mixed on Mar. 27, 2015, Initial pH listed as 7.5 and then after being mixed the pH was 6.73 with my meter.
• Maple tree sap is 2-2.6% sucrose which is 4-5 times too high and hyperosmolar. BTE normally has 0.5% Fructose in it. However goshawk eggs do not do well with Fructose and must have Sucrose to survive.
• Number 2 recipe; Beltsville Turkey Extender, No fructose plus 90 ml Birch tree sap from Alaska, first run sap, QS to 100 ml. dated on bottle Mar. 11, 2015 and mixed on Apr. 11, 2015. Initial pH listed as 7.5 and then after mixing read on my meter as 7.62.
• Number 3 recipe; Beltsville Turkey Extender, No fructose plus 0.5% sucrose, plus 16% Methyl Acetamide by weight. The pH was 7.78. Plus 0.2 mg Inositol (should have been 0.02 mg Inositol). It had a total volume of about 10 ml.
• Number 4 recipe; Beltsville Turkey Extender, No fructose, plus ½% sucrose. The final pH was 7.36. The water was boiled and probably has a low oxygen tension.
• Purdy formulas were a simple addition of maple tree sap, by volume to BTE. The osmolalities were as listed.
• BTE, Control, No sap added, 352 mOsm
• BTE, 5% Maple tree sap, 339 mOsm
• BTE, 10% Maple tree sap, 326 mOsm
• BTE, 20% Maple tree sap, 308 mOsm
• The osmolality of the 20% Maple tree sap was too low to support the cells due to cell swelling.

• TABLE 1
  		Date		Hen	Ratio of	Is this	Glucose	Comments on
  Num-	Date Semen	Semen was	Sire	receiving	Semen:Ex-	sample	Level	Semen Survival
  ber	was Used	collected.	donating.	semen.	tender.	workable?	in mg/dl	and Storage.
  4	Mar. 31,	Apr. 6,	Squirt	Juniper	1 to 2	NO		Less than 1% motile on
  	2014	2011						thaw. Most were dead.
  5	Apr. 11,	Apr. 9,	Squirt	None	1 to 3	NO		Large sample 40-50 ul.
  	2013	2011
  6	Mar. 25,	Apr. 21,	Squirt	None	1 to 2	NO		Some urate
  	2014	2011						contamination. 0% motility
  								on thaw.
  7	X	Apr. 23,	Squirt	None	1 to 2	NO	520	Small amount of urate
  		2011					mg/dl	contamination. 0% motility
  								when thawed. On the
  								blood glucose meter it had
  								a 520 mg/dl reading.
  								Impression; There was
  								decreased speed of
  								motility prefreezing. Was
  								the diluent either too
  								thick?
  8	Apr. 11,	Apr. 11,	Squirt	None	1 to 2	NO		There was a 2 minutes mix
  	2013	2011						time and it went straight to
  								the tank and was
  								immersed. It was thawed
  								on Apr. 11, 2013 and there
  								was 0% motility
  9	X	Apr. 15,	Squirt	None	1 to 4	NO	435	Rare motility upon thaw.
  		2011					mg/dl	Less than 1%.
  10	Apr. 11,	Apr. 16,	Squirt	None	1 to 4	NO		Heavy urate contamination
  	2013	2011						so I diluted with extender
  								so the semen survives.
  								O % motility on thaw.
  11	Mar. 31,	Apr. 16,	Squirt	None	1 to 1	NO		0% motility on thaw.
  	2014	2011
  12	Apr. 11,	Apr. 24,	Squirt		1 to 2	NO		2 cells seen moving.
  	2013	2011						Almost no survivorship
  13	Mar. 31,	Apr. 25,	Squirt	None	1 to 2	NO		0% motility on thaw.
  	2014	2011
  14	Mar. 24,	Apr. 26,	Squirt	Juniper	1 to 2	NO		0% motility on thaw.
  	2013	2011
  15	Mar. 24,	Apr. 26,	Squirt	None	1 to 0	NO	BG 5	2% Motile questionable
  	2013	2011					mg/dl,	with thawing.
  							retested
  							as 66
  							mg/dl
  16	Mar. 31,	Apr. 27,	Squirt	None	1 to 2	NO		0% motility on thaw.
  	2014	2011
  17	Mar. 24,	Apr. 29,	Jasper	None	1 to 2	NO		Dies quickly when viewed
  	2014	2011						as a wet prep w/o
  								freezing. 0% motility upon
  								thawing.
  18	Mar. 26,	Apr. 25,	Squirt	Juniper	Unknown	NO		1-5% Motility on thaw.
  	2014	2011
  19	Mar. 25,	Apr. 30,	Squirt	Juniper	1 to 2	NO		Tail agglutination a
  	2014	2011						problem. Survives 3-4
  								hours at room
  								temperatures. Viewed as a
  								good sized sample wet
  								prep prior to freezing.;
  								Upon thawing saw 5% or
  								less moving and est 1%
  								forward motility. Then put
  								this in Juniper.
  20	Mar. 25,	May 1,	Squirt	None	1 to 3	NO		Viewed prior to freezing,
  	2014	2011						tail agglutination is a
  								problem, there is
  								decreased survivorship
  								after the extender is frozen
  								in the fridge and used after
  								thawing. Dilution also not
  								1 to 2. Viewed as a trace
  								sample wet prep.
  								Exploded on thaw.
  								Capillary tube found and
  								trace saw 0% motility.
  21	Apr. 11,	May 3,	Squirt	None	1 to 2	NO		Good survivorship in
  	2013	2011						diluent w/o freezing.
  								Survived 1:30 PM to 6:50
  								PM, some survival at room
  								temperature. When
  								thawed after freezing there
  								was 0% survival.
  22	Apr. 11,	Apr. 6,	Squirt	None	1 to 3	NO		Some urate
  	2013	2013						contamination. 0% motility
  								on thaw.
  23	Mar. 30,	May 4,	Squirt	Juniper	1 to 2	YES		I put this in Juniper at 1
  	2014	2011						PM. 25% survivorship with
  								good motility upon
  								thawing. A lot of
  								autoagglutination.
  								Extender frozen prior to
  								use in the freezer. Some
  								survival at room
  								temperature 1 PM to
  								6:50 PM with little forward
  								motility in wet prep.
  								Impressions: Trout #1
  								seemed better; Extender
  								best used fresh and not
  								frozen; Cells died faster on
  								this slide and there was
  								more agglutination-Stored
  								extender in 5 cc vials and
  								it was colder at time of
  								use. (Important = Cold
  								shock.)
  24	Apr. 2,	May 14,	Squirt	Juniper	2 to 5	MAYBE		Heavy urate
  	2014	2011						contamination. On thaw
  								there was less than 5%
  								motility due to trauma from
  								the explosion of liquid
  								nitrogen. Kevin retrieved
  								this from the can. Used in
  								Juniper the day the tank
  								was filled.
  25	Jun. 13,	Apr. 10,	Squirt	None	1 to 4	NO		Minor urate contamination,
  	2013	2013						1% good forward motility
  								with a Ice water thaw.
  26	Apr. 11,	Apr. 11,	Squirt	None	1 to 3	NO		Minor urate contamination,
  	2013	2013						0% forward motility on Ice
  								water thaw.
  27	Jun. 2,	Apr. 11,	Squirt	None	1 to 8	NO		0% motility on cold water
  	2013	2013						thaw. No cryoprotectant
  								used.
  28	Jun. 3,	Apr. 12,	Squirt	None	1 to 4	NO		Lost most of the sample.
  	2013	2013						Unable to evaluate. No
  								survival seen in ones
  								diluted with water. Cold
  								water thaw.
  29	Mar. 31,	May 7,	Squirt	Juniper	1 to 3	NO		Less tail agglutination with
  	2014	2011						this formula. More rapid
  								cell death though after
  								only 20 minutes. Maybe
  								30% survival, 5% forward
  								motility at 1 hour. None
  								alive on wet prep at 6:50
  								PM, (collected 1 PM),
  								impression, sperm dies
  								fast in this extender. Less
  								than 1% motile on thaw
  								and put in Juniper
  								Mar. 31, 2014.
  30	Apr. 14,	Apr. 4,	Squirt		1 to 2	NO		No motility seen on fresh
  	2013	2013						wet prep. 7 very mobile
  								cells seen, 1 cell was
  								moving fast and then
  								slowed down and stopped.
  								I used Trout #2 plus
  								¼ tsp Sorbitol and ⅛ tsp
  								Arabogalactin.
  31	Mar. 30,	May 30,	Squirt	Juniper	1 to 4	MAYBE		Trace sample after
  	2014	2013						exploding saw some
  								motility
  32	X 2014	May 31,	Squirt	Juniper	16%	NO		8 mm of Semen placed in
  		2013						room temperature Turkey
  								Extender went to the
  								fridge for 30 minutes
  								acclimation time, Added
  								3 units DMA for a final
  								volume of 50 mm. Quickly
  								placed over liquid nitrogen
  								in under 60 seconds.
  33	X 2014	Jun. 1,	Squirt	Juniper	44%	NO		22 mm of Semen was
  		2013						placed in Turkey Extender
  								at room temperature and
  								placed in the fridge and
  								acclimated for 30 minutes.
  								3 units/ul of DMA was
  								added. I was placed over
  								the liquid nitrogen in under
  								60 seconds. It was hung
  								over the vapors for 10
  								minutes and then flash
  								frozen.
  34	Mar. 29,	Jun. 2,	Squirt	Juniper	48%	NO		24 mm of Semen was
  	2014	2013						placed in Turkey Extender
  								at room temperature to a
  								volume of 47 mm and then
  								acclimated in the fridge for
  								30 minutes. 3 ul of DMA
  								was added quickly and
  								then the sample was hung
  								over the vapor (in less
  								than 60 seconds) for 10
  								minutes and then
  								immersed in liquid
  								nitrogen.
  35	Apr. 3,	May 28,	Squirt	Juniper	20%	NO		8-10 mm of Semen was
  	2014	2013						collected and Turkey
  								Extender was added that
  								was at fridge
  								temperatures. The final
  								volume with DMA was
  								50 ul/mm. 35 mm of
  								Extender and 5 ul of DMA
  								was used to make it 10%
  								DMA. “No motility”
  								(probably too cold) seen
  								on the smear on the fresh
  								wet prep. When thawed on
  								ice water 5-10% were
  								seen moving. Only 1%
  								with good not great
  								forward motility. There was
  								progressive loss of motility
  								over minutes. (45
  								minutes). This is the likely
  								sample that went into
  								Juniper on 4/3.
  36	X 2014	May 29,	Squirt	Juniper	8%			4 mm of Semen was
  		2013						collected and Turkey
  								Extender that was
  								refrigerator temperature
  								was added to a volume of
  								47 mm. This was left in the
  								fridge at 40 F. from 7 AM to
  								6:30 PM, 3 ul of DMA was
  								added quickly and in less
  								than a minute it was hung
  								over the vapors for 10
  								minutes and then
  								immersed. 50% great
  								motility off trace sample
  								seen before adding the
  								DMA.
  37	Mar. 26,	Jun. 3,	Squirt	None	8%			4 mm of Semen was
  	2014	2013						collected and Turkey
  								Extender was added to a
  								volume of 45 mm. It was
  								acclimated for 30 minutes
  								and then 5 ul (10%) DMA
  								was added. It was Flash
  								Frozen.
  38	Mar. 26,	Jun. 3,	Squirt	None	8%	NO		2 mm of Semen was
  	2014	2013						collected and Turkey
  								Extender 23 mm was
  								added and it was
  								acclimated in the fridge for
  								30 minutes. (10%) DMA
  								was added quickly and it
  								was Flash Frozen in under
  								a minute.
  39	Mar. 26,	Jun. 4,	Squirt	None	8%	NO		4 mm of Semen was
  	2014	2013						collected and 45 mm of
  								Turkey Extender was
  								added and it was
  								acclimated in the fridge for
  								30 minutes. 5 ul of DMA
  								was added (10%) and it
  								was Flash Frozen after
  								mixing in under a minute.
  40	Mar. 26,	Jun. 5,	Squirt	None	4%	NO		1 mm of Semen was
  	2014	2013						collected and 8 mm of
  								Turkey Extender was
  								added and it was
  								acclimated in the fridge for
  								30 minutes. 2 ul of DMA
  								was added and it was
  								flash frozen after mixing in
  								under a minute.
  41	2014	2013 4	Squirt	Juniper		YES		4 TUBES THAT WERE
  		SAMPLES						PRESERVED IN 2013
  		FLOATING						THAT WERE PUT IN
  		IN THE						COMMERCIAL PLASTIC
  		LIQUID						TUBES WITH BEADS TO
  		NITOGEN.						CAP THE ENDS, SEMEN
  								WAS IN CAPPILLARY
  								TUBES, WERE FOUND
  								FLOATING IN THE
  								LIQUID NITROGEN ON
  								TOP. BUT LOST THEIR
  								LABELS. SOME OF
  								THESE SAMPLES
  								CONTAINED 25%
  								SURVIVAL OF SEMEN
  								SAMPLES AND WERE
  								ACTIVELY MOVING
  								FORWARD. THESE
  								WERE PUT INTO
  								JUNIPER. ONE OF THE
  								RED AND FOUR OF THE
  								GREEN SAMPLES
  								APPEAR TO BE WHAT I
  								USED FOR AI IN
  								JUNIPER. I DO NOT
  								KNOW WHICH TUBE
  								WAS WHICH BUT I DO
  								KNOW THAT I LOST 3
  								RED SAMPLES ON
  								THAW DUE TO
  								EXPLODING. THE
  								REMAINING SAMPLES
  								DID NOT LEAK IN THE
  								PRIMARY CONTAINER.
  								MOST OF THE GREEN
  								SAMPLES WERE NOT
  								LOST. THE ENTIRE
  								TANK WAS EMPTIED OF
  								ALL OF ITS SAMPLES IN
  								2014. STARTING OVER.
  								16 SAMPLES FOR 2013
  								PUT IN THE TANK.
  42		May 2,	Squirt		3 mm			No urates. Low cellularity.
  		2014			Semen to
  					30 units of
  					Turkey
  					extender,
  					plus 1.5
  					units DMA
  43		May 1,	Squirt		6 mm			Some urate
  		2014			Semen,			contamination. Low
  					diluted to			cellularity.
  					30 mm
  					volume,
  					added 1.5
  					units DMA
  44	Mar. 1,	May 3,	Squirt			NO		No urates. Low cellularity.
  	2016	2014						Mar. 1, 2016 Thawed in an
  								ice water bath. Low
  								cellularity due to males
  								age. Less than 10%
  								motile. Can not do a
  								Live/Dead stain as
  								cellularity is too low.
  45	Mar. 29,	May 4,	Squirt		2 mm of	NO		No urates. Low cellularity.
  	2015	2014			Semen			Goshawk semen requires
  					plus 18 mm			sucrose and not fructose
  					of Turkey			to survive freezing!!! This
  					extender to			is why these cells survived
  					a total			in the Trout Extender #2
  					volume of			and not the Turkey
  					20 mm.			Extender that has the
  								Fructose! You must use
  								Beltsville Turkey Extender
  								minus the Fructose, with
  								sucrose added back in to
  								.5% (½%)
  46		May 4,	Squirt		6 mm of			Low number of urates.
  		2014			Semen,			Low cellularity.
  					plus 19 mm
  					of Turkey
  					Extender,
  					plus 2 ul of
  					DMA.
  47		May 5,	Squirt		1-40 mm			1 side of the split sample
  		2014			sample			had more urates than the
  					contaminated			other, semen in only one
  					with			spot on the tube, so
  					urates split			separated into ½ to put
  					into 2-20			most urates in 1 tube. Low
  					mm			cellularity.
  					samples,
  					plus 28 mm
  					Turkey
  					extender,
  					plus 3 ul of
  					DMA (6%).
  48		May 6,	Squirt		6 mm of			No urates. Low cellularity.
  		2014			Semen,
  					plus 14 mm
  					of Turkey
  					extender to
  					a total
  					volume of
  					20 mm.
  					Plus 1.5 ul
  					of DMA,
  					(7%)
  49		May 6,	Squirt		10-16 mm			Many urates. Small
  		2014			of Semen			amount of semen in one
  					plus 47 mm			spot. Final volume 50 mm.
  					of Turkey			Low cellularity.
  					extender,
  					plus 3 ul of
  					DMA.
  50		May 7,	Squirt		6 mm of			No urates. Low cellularity.
  		2014			semen plus
  					19 mm of
  					Turkey
  					extender
  					plus 2 ul of
  					DMA.
  51		May 7,	Squirt		6 mm of			No urates. Low cellularity.
  		2014			Semen			Lots of blast cells seen.
  					plus 19 mm			Goshawk semen requires
  					of Turkey			sucrose and not fructose
  					extender			to survive freezing!!! This
  					plus 1.25 ul			is why these cells survived
  					of DMA			in the Trout Extender #2
  					(5%).			and not the Turkey
  								Extender that has the
  								Fructose! You must use
  								Beltsville Turkey Extender
  								minus the Fructose, with
  								sucrose added back in to
  								.5% (½%)
  52	Mar. 29,	May 9,	Squirt	None	5 plus mm	NO		Watery urates seen in 8 of
  	2015	2014			of Semen			13 mm total initial semen
  					and about			volume so had about 5
  					8 mm of			mm of semen present.
  					Urates,			Low cellularity. Goshawk
  					plus 27 mm			semen requires sucrose
  					of Turkey			and not fructose to
  					extender to			survive!! These cells can
  					a total			not use fructose and this is
  					volume of			why these cells survive in
  					40 mm plus			the Trout #2 extender!
  					2.7 ul of
  					DMA (6%)
  53		May 10,	Squirt		16 mm of			No urates and cellularity is
  		2014			Semen			low but going up.
  					plus 22 mm
  					of Turkey
  					extender
  					for a final
  					volume of
  					38 mm,
  					plus 2.23 ul
  					of DMA,
  					(6%).
  54		May 22,	Squirt		5 mm of			No urates and cellularity is
  		2014			Semen			low but going up.
  					plus 15 mm
  					of Turkey
  					extender to
  					a final
  					volume of 2
  					mm, plus
  					1 ul of DMA
  					(5%).
  55		May 12,	Squirt		14 mm of			A few urates, but not a lot.
  		2014			Semen			Low cellularity but
  					plus 23 mm			increasing.
  					Turkey
  					extender to
  					a total of 47
  					mm. Plus
  					2.5 ul of
  					DMA (5%).
  56	May 14,	May 13,	Squirt	None	4 mm of	No	Unknown,	No urates. Cellularity is
  	2014	2014			Semen		meter	low but climbing.
  					plus 20 mm		could not	Storage straw did not leak
  					of Turkey		read this	and the cell survivorship
  					extender		number.	was feeble.
  					plus 5%
  					Maple
  					Syrup, plus
  					1 ul DMA
  					(5%).
  57	Apr. 4,	Mar. 22,	Odin	None	8 mm of	No		No semen survived
  	2015	2015			Semen			freezing. The fructose in
  					plus 11 ul			the sample is the
  					of Beltsville			suspected problem
  					Turkey			because it slows the
  					Extender			speed of the spermatozoa
  					(unaltered)			down by half in fresh
  					Plus 1 ul of			samples extended with
  					DMA. Total			this TE. The
  					volume			cryoprotectant needs to
  					20 ul.			also be looked at. All
  								2015 samples placed in
  								cold Eppendorf tubes that
  								were already in the fridge.
  								Temperature shock might
  								be present. Might be too
  								cold next to refrigerator
  								coils. Goshawk semen
  								requires sucrose and not
  								fructose to survive
  								freezing!!! This is why
  								these cells survived in the
  								Trout Extender #2 and not
  								the Turkey Extender that
  								has the Fructose! You
  								must use Beltsville Turkey
  								Extender minus the
  								Fructose, with sucrose
  								added back in to .5%
  								(½%)
  58	Apr. 18,	Mar. 23,	Odin	None	30 ul of	NO		All 2015 samples placed in
  	2015	2015			Semen			cold Eppendorf tubes that
  					plus 30 ul			were already in the fridge.
  					of Turkey			Temperature shock might
  					extender			be present. Might be too
  					plus			cold next to refrigerator
  					Inositol.			coils. Lost sample across
  					Plus 2.6 ul			the garage as it exploded.
  					of DMA,
  					62.6 ul total
  					volume.
  59	Apr. 18,	Mar. 23,	Odin	None	30 ul of	NO		A sample that was still left
  	2015	2015			Semen			in the tube before freezing
  					plus 30 ul			had 25% survival based
  					of Turkey			on a live/dead stain. All
  					extender			2015 samples placed in
  					plus			cold Eppendorf tubes that
  					Inositol.			were already in the fridge.
  					Plus 2.6 ul			Temperature shock might
  					of DMA.			be present. Might be too
  					62.6 ul total			cold next to refrigerator
  					volume. So			coils. Less than 1%
  					this is 4%			Motile with 55 F. water bath
  					DMA.			and hand warming.
  60	Apr. 18,	Mar. 25,	Odin	None	12 ul of	NO		All 2015 samples placed in
  	2015	2015			Semen			cold Eppendorf tubes that
  					plus 24 ul			were already in the fridge.
  					of Turkey			Temperature shock might
  					Extender			be present. Might be too
  					plus			cold next to refrigerator
  					Inositol.			coils. No survival on
  					Plus 2 ul of			thawing. Thawed in a cool
  					DMA. 38 ul			water bath at 55 F. and
  					total			hand warming.
  					volume. So
  					this is 5%
  					DMA
  61	Apr. 18,	Mar. 25,	Odin	None	10 ul of	NO		All 2015 samples placed in
  	2015	2015			semen plus			cold Eppendorf tubes that
  					18 ul of			were already in the fridge.
  					Turkey			Temperature shock might
  					extender			be present. Might be too
  					plus			cold next to refrigerator
  					Inositol,			coils. Thawed in cool
  					plus 2 ul of			water bath at 55 F. and
  					DMA To a			hand warming. No cell
  					total			survival.
  					volume of
  					30 ul. 6.6%
  					DMA
  62	Apr. 15,	Mar. 27,	Odin	None	15 ul of	NO		All 2015 samples placed in
  	2015	2015			Semen			cold Eppendorf tubes that
  					plus 30 ul			were already in the fridge.
  					of Beltsville			Temperature shock might
  					Turkey			be present. Might be too
  					Extender			cold next to refrigerator
  					plus			coils. Thawed in cool
  					Inositol,			water bath at 55 F. and
  					plus 2 ul of			then put on a warming
  					DMA, or			plate. Less than 1%
  					4.25%			survival.
  					DMA
  63	Apr. 15,	Mar. 27,	Odin	None	20 ul of	NO		All 2015 samples placed in
  	2015	2015			Semen			cold Eppendorf tubes that
  					plus 37 ul			were already in the fridge.
  					of Beltsville			Temperature shock might
  					Turkey			be present. Might be too
  					Extender			cold next to refrigerator
  					plus			coils. Thawed in a cool
  					Inositol,			water bath at 55 F. No
  					plus 3 ul of			cells survived.
  					DMA, 5%
  					DMA
  64	Apr. 15,	Mar. 28,	Odin	None	16 ul of	NO		All 2015 samples placed in
  	2015	2015			Semen			cold Eppendorf tubes that
  					plus 29 ul			were already in the fridge.
  					of Beltsville			Temperature shock might
  					Turkey			be present. Might be too
  					Extender			cold next to refrigerator
  					with			coils. Thawed in a cool
  					Inositol,			water bath at 55 F. No
  					plus 3 ul of			cells survived.
  					DMA,
  					6.25%
  					DMA
  65	Apr. 15,	Mar. 28,	Odin	None	14 ul of	NO		All 2015 samples placed in
  	2015	2015			Semen in			cold Eppendorf tubes that
  					16 ul of			were already in the fridge.
  					Beltsville			Temperature shock might
  					Turkey			be present. Might be too
  					Extender			cold next to refrigerator
  					plus			coils. Thawed in a cool
  					Inositol,			water bath at 55 F. and
  					plus 2 ul of			then put on a warming
  					DMA,			plate. No cells survived.
  					6.25%
  					DMA
  66	Apr. 15,	Mar. 28,	Odin	None	13 ul of	NO		All 2015 samples placed in
  	2015	2015			Semen in			cold Eppendorf tubes that
  					20 ul of			were already in the fridge.
  					Beltsville			Temperature shock might
  					Turkey			be present. Might be too
  					Extender			cold next to refrigerator
  					plus			coils.
  					Inositol,
  					plus 2 ul of
  					DMA, 5.7%
  					DMA.
  67	Mar. 30,	Mar. 29,	Odin	None	16 ul of	MAYBE	5 times	This sample was flash
  	2015	2015			Semen		higher	frozen with no acclimation.
  					plus 24 ul		than BTE	When I thawed it, it took
  					of BTE, no		normally	off living just like the
  					fructose,		is so this	sample did that was
  					plus Maple		is	viewed at room
  					tree sap,		hyper-	temperature. There was
  					no other		osmolar.	about 11% motility and
  					cryoprotectant			less than half of these
  								were moving forward well
  								on thaw. A sample that
  								was left on a slide at room
  								temperature had the
  								mature spermatids stop
  								moving within 5 minutes
  								but the immature
  								spermatids did well and
  								kept on moving. The
  								speed of movement was
  								much better and this is
  								clearly an improvement
  								over the BTE with
  								fructose. I can assume
  								that goshawk semen
  								needs sucrose to survive.
  								The sap is 100
  								milliosmoles and was
  								added to the dry
  								ingredients of the BTE, no
  								fructose. This added in
  								sucrose at 5 times the
  								percentage needed and
  								made it hyperosmolar. The
  								caused the mature cells to
  								die. The immature
  								spermatids, with immature
  								cell walls, could equalize
  								the osmotic pressure.
  								Maple sap is .5-2.6%
  								sucrose. This is first run
  								sap so it is higher in
  								sucrose than last run.
  								Different maple tree
  								species have different
  								percentages of sucrose.
  68	Apr. 15,	Mar. 29,	Odin	None	?	NO		6% DMA, No survival
  	2015	2015
  69	Mar. 31,	Mar. 29,	Odin	None	19 ul of	MAYBE,	Sucrose	Most of the sample was
  	2015	2015			Semen	MORE	likely 5	lost on thaw. The trace in
  					plus 38 ul	SUCCESS	times	the sample has 66%
  					of BTE plus	THAN I	what is	motility based on visual
  					Maple tree	HAD	needed,	estimates. 25 out of 43
  					sap, first	HOPED	so this	alive on a swim count with
  					run, tree	FOR.	sample is	the counter. On a live
  					number 3.		hyper-	dead stain there was Live
  					No other		osmolar.	58/42 Dead!
  					cryoprotectant
  70	Apr. 15,	Apr. 3,	Odin	None	25 ul of	YES, but	Sucrose	This tube is completely
  	2015	2015			Semen,	can do	likely 5	full. 5-10% motility on thaw
  					plus BTE,	better.	times	with normal motility.
  					no fructose,		what is	Thawed in 55 F. water bath
  					plus 50 ul		needed,	and then placed on a
  					of Maple		so this	warm plate.
  					tree sap,		sample is
  					No other		hyper-
  					cryoprotectant.		osmolar.
  71	Apr. 15,	Apr. 3,	Odin	None	16 ul of	YES	Sucrose	Thawed in 55 F. water bath
  	2015	2015			Semen		likely 5	and then placed on a
  					plus BTE,		times	warming plate. 10%
  					no fructose,		what is	motility at thaw and then
  					plus 28 ul		needed,	the cells slow their motility
  					of Maple		so this	to 5% estimated visually
  					tree sap,		sample is	over 5 minutes.
  					No other		hyper-
  					cryoprotectant.		osmolar.
  72	Apr. 15,	Apr. 4,	Odin	None	13 ul of	YES	Sucrose	Thawed in a 55 F. water
  	2015	2015			Semen		likely 5	bath and then a warming
  					plus BTE,		times	plate. 20% near normal
  					no fructose,		what is	motility and motility
  					plus 26 ul		needed,	estimated visually.
  					of Maple		so this	Thawed fast! Ventilated
  					Tree sap,		sample is	soda straw is key.
  					no other		hyper-
  					cryoprotectant		osmolar.
  					was
  					used.
  73	Apr. 15,	Apr. 12,	Odin	None	20 ul of	NO	Sucrose	At room temperature, cells
  	2015	2015			Semen		level	slow rapidly on a wet prep,
  					plus 40 ul		reads at	probably still too
  					of BIRCH		479 mg/dl	hyperosmolar. No motility
  					tree sap		on a	on thaw with 55 F. water
  							Accucheck	bath and then a warming
  							glucose	plate.
  							meter.
  74	Apr. 14,	Apr. 12,	Odin	NONE	42 ul of	MAYBE		Apr. 14 2015 Sample #1
  	2015	2015			Semen,			Orange straw; Thawed in
  					plus BTE,			cold water and then
  					no fructose,			placed on a warming
  					plus			plate. 5-10% motility of
  					Sucrose,			normal looking sperm. The
  					Added in			rest are moving a little but
  					75 ul of			not swimming forward.
  					BTE, minus
  					fructose,
  					plus
  					sucrose ½%
  					plus MA
  					(methyacetamide)
  75		Apr. 13,	Odin		45 ul of
  		2015			Semen split
  					between 3
  					straws,
  					BTE no
  					fructose,
  					plus ½%
  					sucrose,
  					plus 84 ul
  					of BTE
  					(same as
  					above) with
  					MA
  					(Methyacetamide)
  76	Feb. 28,	Apr. 14,	Odin	None	7 ul of	Yes		Sample was prepped at
  	2016	2015			Semen			room temperature with
  					with 26 ul			all items starting at 70 F.
  					of BTE, no			Feb. 28, 2016 Teal Blue
  					fructose,			Straw, Not ventilated,
  					plus ½%			80% of cells vibrating,
  					sucrose.			10% moving actively,
  					Plus 2 ul			Thawed in a 41 F. water
  					of DMA			bath. Live 65/45 Dead
  					cryoprotectant.			Stain.
  					Final
  					volume of
  					35 ul.
  77	Feb. 28,	Apr. 14,	Odin	None	55 ul of	This is		All materials start at room
  	2016,	2015			Semen	one of the		temperature (70 F.) and
  	Mar. 1,				plus 110 ul	first		then go to fridge to chill
  	2016				of BTE, no	samples		to 41 F. Cold packs were
  					fructose,	of NO		used to carry to the
  					plus ½%	FRUCTOSE		garage.
  					sucrose,	plus		Feb. 28, 2016 Thawed in
  					chilled in	DMA to		41 F. water bath to a
  					the fridge	test if it is		warming plate with Live
  					for 10	the		39/61 Dead.
  					minutes,	fructose		Mar. 1, 2016 Thawed in a
  					plus 8 ul of	or DMA		41 F. water bath to a
  					DMA	that is		warming plate with 10%
  						causing		swimming normally and
  						the		50% vibrating in place.
  						samples
  						to fail.
  						Feb. 28, 2016
  						Yes, No
  						movement-
  						green
  						tube,
  						Thawed
  						41 F.
  						water
  						bath.
  						Little cell
  						deformity
  						with a
  						Live
  						39/61
  						Dead
  						stain.
  						Sample
  						quiescent.
  						Mar. 1, 2016
  						Yes, 10%
  						are
  						swimming
  						normally,
  						50% are
  						vibrating,
  						Thawed
  						in a 41 F.
  						water
  						bath. Live
  						32/68
  						Dead
  						stain.
  78	Apr. 18,	Apr. 15,	Odin	None	38 ul of	Maybe,		Acclimated at 41 F. in
  	2015,	2015			Semen,	because		fridge with sap separate
  	Apr. 18,				plus 75 ul	the sap		from semen until placed in
  	2015				of Birch	preserves		capillary tubes for
  					Sap, BTE,	the		freezing. Acclimated for 10
  					no fructose,	semen		minutes.
  					first run..	when		Apr. 18, 2015 Thawed in a 55
  						frozen.		F. water bath. Sample 1
  						But the		showed 5% spermatozoa
  						amount of		moving forward normally
  						sap		with a live/dead stain of
  						needs to		32/68.
  						be		May 18, 2015 Sample 2 had
  						reduced.		about 5% moving forward
  						Similar		normally with a live/dead
  						response		stain of 34/66.
  						to Maple
  						tree sap.
  						The cells
  						survive
  						the freeze
  						but stop
  						moving
  						due to
  						hyperosmolality.
  79	Apr. 18,	Apr. 17,	Odin	None	28 ul of	Maybe,		This was first run Birch
  	2015,	2015			Semen,	because		tree sap from Alaska. It
  	Apr. 18,				plus 28 ul	the sap		was acclimated after
  	2015				BTE, no	preserves		mixed by only placing it
  					fructose,	the		between gel packs that
  					plus ½%	semen		were at 41 F. from the
  					sucrose,	when		fridge and then it was flash
  					plus 56 ul	frozen,		frozen. Two straws were
  					Birch Tree	but the		made from this sample.
  					sap in BTE,	amount of		One was a yellow, and the
  					no fructose,	sap		other green, ventilated
  					final	needs to		soda straws. One tube
  					volume of	be		had 2-5% forward moving
  					112 ul with	reduced.		sperm on visual estimate
  					1:3 semen	Similar		with a live/dead stain of
  					to Birch	response		21/79. The second tube
  					tree sap	to Maple		had 0% forwardly moving
  					extender.	tree sap.		and no live dead stain was
  						The cells		done. Thawed in a 55 F.
  						survive		cool water bath.
  						the freeze
  						but lose
  						motility
  						due to
  						hyperosmolality.
  80	Apr. 18,	Apr. 17,	Odin	None	50 ul of	Maybe,		Thawed in a cool water
  	2015,	2015			Semen had	Maybe		bath of 55 F.
  	Apr. 18,				50 ul of			Apr. 18, 2015 First sample 2-3%
  	2015				BTE, no			normal motility, with a
  					fructose,			live/dead stain of 57/43.;
  					plus 1/%			Apr. 18, 2015The second
  					sucrose			sample had 5-10%
  					added			moving forward normally
  					together in			and a live dead stain of
  					1 tube.			42/58.
  					Later 100
  					ul of BTE,
  					no fructose,
  					plus Maple
  					sap was
  					added for a
  					final
  					volume of
  					200 ul.
  					Making the
  					semen ¼
  					of the total
  					mix.
  81	Apr. 18,	Apr. 18,	Odin	None	22 ul of	YES,		Thawed in a cool water
  	2015,	2015			Semen had	YES		bath of 55 F.
  	Apr. 18,				22 ul of			Apr. 18, 2015 First sample
  	2015				BTE, no			had 25-30% normal
  					fructose,			motility with a live dead
  					plus ½%			stain of 50/50.;
  					sucrose			Apr. 18, 2015 The second
  					added to it			sample had about 55%
  					placed in 1			normal forward motility
  					tube. Later			with a live/dead stain of
  					44 ul of			57/43. 100 cells were
  					BTE, no			counted in each group.
  					fructose,
  					plus Maple
  					tree sap
  					was added
  					for a final
  					volume of
  					88 ul.
  82	May 10,	Apr. 20,	Odin	None	65 ul of	Yes,		Acclimated at 41 F. in
  	2015,	2015			Semen,	Maybe,		fridge with sap separate
  	Feb. 24,				plus 65 ul	No, No		from semen until placed in
  	2016,				BTE minus			capillary tubes for
  	Green				fructose,			freezing. Acclimated for 10
  	straw-				plus ½%			minutes. Then flash
  	Holder 5,				sucrose.			frozen. Ice water thaw,
  	Mar. 2,				Later 130			10-15% good motility, Live
  	2016				ul of BTE			Dead Stain 24 live/76
  	Green				minus			dead.
  	straw-				fructose			Feb. 24, 2016 Thawed in a 41
  	Holder 6,				plus Birch			F. ice water bath, 2-3%
  	Mar. 2,				tree sap			moving forward well, pH
  	2016				first run.			7.0-7.2 with a Live22/78
  	Green							Dead Stain.
  	straw							Mar. 2, 2016 Thawed in a
  	Holder 6.							41 F. Ice water bath, Green
  								Straw Holder 6, Rare
  								motile sperm with a Live
  								10/90 Dead Stain. Cells
  								are very distorted.
  								Mar. 2, 2016 Thawed in an
  								ice water bath at 41 F. No
  								motility and the cells are
  								very deformed. Live 7/93
  								Dead.
  83	May 3,	Apr. 20,	Odin	None	60 ul of	No, No		Acclimated at 41 F. in
  	2015,	2015			Semen,	Mar. 6, 2016		fridge with sap separate
  	Mar. 6,				plus 60 ul	Yellow		from semen until placed in
  	2016,				of BTE −	straw,		capillary tubes for
  	Mar. 6,				fructose, +	water got		freezing. Acclimated for 10
  	2016				½%	into		minutes. Then flash
  					sucrose.	sample.		frozen.
  					Later 60 ul	YES		May 3, 2016 Thawed in Ice
  					of BTE plus	Mar. 6, 2016		water, 2% forward
  					Maple tree	Yellow		motility, Live dead stain,
  					sap, first	straw		39 live/61 dead.
  					run Tree			Mar. 6, 2016 Thawed in ice
  					#3. added			water at 41 F. 1% Motile
  					for a final			and 1% moving in place.
  					volume of			Live 33/67 Dead stain. A
  					180 ul.			lot of agglutination. It was
  								exposed to water on thaw
  								because it lost the caulk
  								on the end of the tube.
  								Mar. 6, 2016 Thawed in a ice
  								water bath. 15-20% are
  								motile. Agglutination is
  								present. Live 49/51 Dead.
  84	May 3,	Apr. 21,	Odin	None	50 ul of	YES,		Acclimated at 44 F. in
  	2015,	2015			Semen had	YES,		fridge with sap separate
  	Mar. 1,				50 ul of	YES		from semen until placed in
  	2016,				BTE, no			capillary tubes for
  	Mar. 6,				fructose,			freezing. Acclimated 10
  	2016				plus 1/%			minutes. Then flash
  					sucrose			frozen.
  					added			May 3, 2015 Thawed in Ice
  					together in			water bath, 55%-60%
  					1 tube.			forward motility, Live dead
  					Later 100			stain 39/61. No pH done.
  					ul of BTE,			Mar. 1, 2016 Thawed in an
  					no fructose,			ice water bath at 41 F.,
  					plus Maple			Over half are moving
  					sap was			forward with fast motility,
  					added for a			pH of 7, with a Live 64/36
  					final			Dead stain.
  					volume of			Mar. 6, 2016 Thawed in an
  					200 ul.			ice water bath at 41 F.
  					Making the			Over 60% are motile with
  					semen ¼			little deformity. They have
  					of the total			fast motility with a Live
  					mix.			73/27 Dead stain.
  85	Mar. 2,	Apr. 21,	Odin	None	50 ul of	Maybe -		Acclimated at 44 F. in
  	2016,	2015			Semen had	Quiescent,		fridge with sap separate
  	Mar. 2,				50 ul of	Maybe		from semen until placed in
  	2016				BTE, no	Quiescent		capillary tubes for
  					fructose,			freezing. Acclimated 10
  					plus 1/%			minutes. Then flash
  					sucrose			frozen.
  					added			Mar. 2, 2016 Thawed in an
  					together in			ice water bath and put on
  					1 tube.			a warming plate. Had 5%
  					Later 50 ul			forward motility and a Live
  					of BTE, no			36/64 Dead stain.
  					fructose,			Mar. 2, 2016 Yellow straw,
  					plus Maple			Thawed in an ice water
  					sap was			bath at 41 F. Less than 1%
  					added for a			are motile with a Live
  					final			40/60 Dead stain.
  					volume of
  					150 ul.
  					Making the
  					semen ⅓
  					of the total
  					mix.
  86	May 3,	Apr. 21,	Odin	None	10 ul of	No,		No acclimation in the
  	2015,	2015			semen plus	Maybe		fridge. Flash frozen.
  	May 3,				10 ul of			May 3, 2015 First sample
  	2015				BTE − Fruc, +			thawed in ice water, No
  					½%			motility, pH of 7 on pH
  					sucrose,			paper, May 3, 2015 Second
  					PLUS			sample thawed in ice
  					YOLK,			water, 2-5% motility, pH 7
  					Later 12 ul			on paper. Live dead stain
  					of BTE −			48 live/52 dead.
  					Fruc, Plus
  					Maple sap,
  					first run
  					tree 3.
  87	Mar. 7,	Apr. 22,	Odin	None	16 ul of	Maybe -		Acclimated in the fridge in
  	2016	2015			Semen,	Quiescent.		separate tubes at 43 F.
  					plus 16 ul	The		Mixed, packaged, and
  					BTE − Fruc, +	cells are		then flash frozen.
  					½%	not		Mar. 7, 2016 Pink soda straw
  					sucrose,	distorted		1% Motile/Cells not
  					PLUS	but likely		distorted. Live 19/81 Dead
  					YOLK,	quiescent		stain.
  					Later 16 ul	and not
  					BTE − Fruc,	motile.
  					plus Maple
  					sap, plus
  					10% Yolk.
  					Final
  					volume
  					48 ul.
  88	May 3,	Apr. 22,	Odin	None	53 ul of	Yes, No		Acclimated in the fridge in
  	2015,	2015			Semen,	Thawing		separate tubes at 43 F.
  	Mar. 2,				plus 53 ul	too warm.		Mixed, packaged, and
  	2016				of BTE −	No Cold		then flash frozen.
  	Pink				fructose, +	water		May 3, 2015 Thawed in Ice
  	straw				½%	thaw is no		water, 10% moving
  	Holder				sucrose,	better.		forward, pH of 7. Live
  	#6,				PLUS			dead stain, Live 38/62
  	Mar. 6,				YOLK,			Dead.
  	2016				Later 53 ul			Mar. 2, 2016 Thawed in a
  	Pink				of BTE −			55 F. water bath, Less than
  	straw				fructose, +			1% motile, many cells
  	Holder				Maple sap,			deformed with a Live
  	#6.				plus 10%			16/84 Dead stain.
  					YOLK			Mar. 6, 2016 Thawed in an
  								ice water bath, 0% motile,
  								Cells not deformed with a
  								Live 13/87 Dead stain.
  89	May 10,	Apr. 23,	Odin	None	45 ul of	No, No		Acclimated in the fridge in
  	2015,	2015			Semen with			separate tubes at 43 F.
  	Mar. 2,				55 ul of			Mixed, packaged, and
  	2016				BTE −			then flash frozen.
  					fructose, +			May 10, 2015 A trace sample
  					½%			of this tube survived well
  					sucrose			at room temperature. pH
  					and YOLK			of 7, Ice water bath thaw,
  					added in at			Less than 1% motile, Live
  					10%, Later			dead stain could not be
  					35 ul of			done. Could not read the
  					BTE +			Live/Dead stain due to the
  					Birch Tree			yolk being present in the
  					Sap so that			sample.
  					it becomes			Mar. 2, 2016 Orange tube,
  					26% Birch			Thawed in an ice water
  					Tree Sap.			bath, Less than 1% motile
  								with a Live 33/67 Dead
  								stain. Many cells are
  								deformed.
  90	May 3,	Apr. 23,	Odin	None	45 ul of	Maybe,		Acclimated in the fridge in
  	2015,	2015			Semen with	Maybe		separate tubes at 43 F.
  	Mar. 1,				55 ul of	appears		Mixed, packaged, and
  	2016,				BTE −	Quiescent.		then flash frozen.
  	Mar. 2,				fructose, +	Maybe		May 3, 2015 Thawed in a
  	2016				½%	appears		55 F. water bath. Less than
  	Holder 6				sucrose	Quiescent.		1% motile or no motility.
  	Green				and YOLK			Mar. 11, 2016 Thawed in a ice
  	Straw				added in at			water bath, No motility but
  					10%, Later			the cells are not deformed
  					32 ul of			with a Live 34/66 Dead
  					BTE +			stain = Quiescence.
  					Birch Tree			Mar. 2, 2016 Thawed in a ice
  					Sap so that			water bath. No motility but
  					it becomes			the cells are not deformed
  					19.7%			with a Live 31/69 Dead
  					Birch Tree			stain = Quiescence.
  					Sap.
  91	Feb. 24,	Apr. 24,	Odin	None	30 ul of	Maybe,		Mixed at room
  	2016	2015			Semen,	Maybe		temperature and then
  	Green				with 60 ul	appears		placed between gel packs
  	soda				of BTE −	Quiescent.		at 43 F. Then flash frozen.
  	straw				fructose, +			Feb. 24 2016 Green soda
  	from				½%			straw from Holder #4
  	Holder				sucrose.			Thawed in an ice water
  	#4,				Later 30 ul			bath at 41 F. Cells not
  	Feb. 24,				of BTE −			deformed and 2-3%
  	2016				fructose,			motile with a Live 23/77
  	Deep				plus ½%			Dead stain.
  	blue				sucrose			Feb. 24 2016 Deep blue
  	soda				plus 18%			straw from Holder #4,
  	straw				MA (diluted			Thawed in an ice water
  	from				to a total			bath at 41 F., Cells not
  	Holder				percentage			deformed 80% and 1%
  	#4.				of 4.5%			motile. But this tube had
  					MA)			10-20% deformed cells
  								overall with a Live 35/65
  								Dead stain.
  92	Feb. 28,	Apr. 25,	Odin	None	15 ul of	Maybe		Mixed at room
  	2016	2015			Semen	appears		temperature and then
  					plus 15 ul	Quiescent		placed between gel packs
  					of BTE −	with 10%		at 43 F. The flash frozen.
  					fructose, +	movement.		Feb. 28, 2016 Thawed in an
  					½%			ice water bath at 41 F.,
  					sucrose,			ventilated soda straw with
  					plus 15 ul			10% moving forward and a
  					of BTE −			Live 35/65 Dead stain.
  					fructose, +			Little cell distortion.
  					½%
  					sucrose
  					plus 18%
  					MA (final of
  					6% MA).
  93	Feb. 24	Apr. 24,	Odin	None	45 ul of	Yes, Yes,		Chilled 10 minutes at
  	2016,	2015			Semen,	Yes		45 F. Mixed packaged
  	Green				with 45 ul			and then flash frozen.
  	straw				of BTE −			Feb. 24 2016 Green straw
  	Holder				fructose, +			Holder #4 Thawed in an
  	#4,				½%			ice water bath at 41 F.,
  	Feb. 24,				sucrose,			cells not deformed with
  	2016				Later 45 ul			2-3% motile. Quiescent
  	Green				of BTE, −			but alive with Live 55/45
  	straw				fructose +			Dead stain.
  	Holder				½%			Feb. 24 2016 Green soda
  	#4,				sucrose			straw Holder #4 Thawed in
  	Feb. 24,				plus 18%			an ice water bath at 41 F.,
  	2016				MA = 6%			cells not deformed, 25-
  	Green				MA final			30% motile and showed
  	straw				concentration			slowing motility over 5
  	Holder							minutes with a Live 57/43
  	#4.							Dead stain.
  								Feb. 24 2016 Green soda
  								straw from Holder #4.
  								Thawed in an ice water
  								bath at 41 F. Cells were
  								not deformed and had 5%
  								motile with slowing of
  								motility over 5 minutes.
  								The live/dead stain was
  								hard to read but had
  								Live18/82 Dead.
  94	May 10,	Apr. 25,	Odin	None	20 ul of	No		Chilled for 10 minutes and
  	2015,	2015			Semen			then added DMA. Ice
  	May 10,				plus 55 ul			water bath thaw. Almost
  	2015				of BTE −			no motility, No live dead
  					fructose, +			stain done. Assessment;
  					½%			DMA is not working.
  					sucrose.
  					Later 4.9 ul
  					of DMA
  					added.
  95	May 3,	Apr. 26,	Odin	None	22 ul of	No,		I AM STARTING THE
  	2015,	2015			Semen	Maybe		PURDY FORMULAS
  	Mar. 6,				plus 66 ul			THAT ARE BTE WITH
  	2016				of BTE +			DIFFERENT
  	Orange				10% Maple			PERCENTAGES OF
  	straw				Sap			MAPLE TREE SAP IN
  					PURDY			THE DILUTION. THESE
  					FORMULA.			HAVE FRUCTOSE AND
  								SUCROSE IN THEM. SEE
  								SHEET ON THESE
  								FORMULAS.
  								May 3, 2016 Ice water bath
  								thaw, 2% moving forward,
  								pH of 7 on paper.
  								Mar. 6, 2016 Thawed in an
  								ice water bath at 41 F.
  								Orange straw with 2%
  								moving forward and 2%
  								moving in place with a
  								Live 14/86 Dead stain.
  96	May 10,	Apr. 27,	Odin	None	18 ul of	No		May 10, 2016 Ice water bath
  	2015	2015			Semen with			thaw, 1-3% of these
  					54 ul of			moving forward. Live dead
  					Purdy 10%			stain 31 live/69 dead.
  					Maple tree			Comments; Fructose, No
  					sap. 1:3			acclimation, and high pH
  					dilutions.			might be a problem.
  97	Mar. 6,	Apr. 27,	Odin	None	12 ul of	YES!		Mar. 6, 2016 No acclimation
  	2016	2015			Semen			and was thawed in an ice
  	Pink soda				plus 36 ul			water bath. Greater than
  	straw				of Purdy 10			50% moving, with 20%
  	Holder #6				% Maple			moving normally. There
  					Tree sap.			was a Live 52/48 Dead
  								stain.
  98		Purdy			Purdy			Purdy formulas begin with
  		Formulas			formulas			#95.
  					begin with
  					#95
  99	Feb. 24,	Apr. 27,	Odin	None	50 ul of	Maybe		Processed at room
  	2016	2015			Semen with	sap		temperature, placed
  	Holder #5				100 ul of	appears		between 43 F. gel packs,
  	Pink soda				Purdy 10%	to		and then flash frozen.
  	straw,				Maple tree	decrease		Feb. 24, 2016 Pink straw
  	Feb. 24,				sap.	movement =		Holder #5 Thawed in an
  	2016					quiescence.		ice water bath at 41 F.,
  	Holder #4					Maybe =		No movement, no pH done,
  	Pink soda					Same,		Cells very distorted. Had a
  	straw,					Maybe =		Live27/73 Dead stain.
  	Feb. 24,					Same.		Feb. 24, 2016 Pink soda straw
  	2016							Holder #4 Thawed in an
  	Holder #4							ice water bath at 41 F.
  	Pink soda							Almost no movement and
  	straw.							cells very distorted. Had a
  								Live 29/71 Dead stain.
  								Feb. 24, 2016 Pink soda straw
  								Holder #4 Thawed in ice
  								water bath at 41 F. Less
  								than 1% moving forward
  								and cells were distorted.
  								Live 36/64 Dead.
  100	May 16,	Apr. 28,	Odin	None	70 ul of	Maybe		Processed at room
  	2015,	2015			Semen	Quiescent,		temperature, placed
  	Mar. 6,				plus 210 ul	Maybe		between 43 F. gel packs,
  	2016,				of Purdy	Quiescent,		and then flash frozen.
  	Mar. 7,				BTE + 20%	No, No		May 16, 2015 Thawed in ice
  	2016				Maple tree			water, 1-5% moving well
  	Green				sap 308			but motility slows quickly.
  	straw from				mOsm.			Live dead stain shows 39
  	Holder #6,							live/61 dead, 30 live/70
  	Mar. 13,							dead. Hot plate on
  	2016							microscope appears to
  	Green							speed loss of motility.
  	straw from							Mar. 6, 2016 Thawed in an
  	Holder #6.							ice water bath at 41 F.
  								10-15% motility swimming
  								forward. Many of the cells
  								are distorted due to the
  								low osmolality. There is a
  								Live 17/83 Dead stain.
  								Mar. 7, 2016 Green straw.
  								Thawed in an ice water
  								bath. Had 1-2% moving.
  								The cells are very
  								distorted and it had a Live
  								20/80 Dead stain.
  								Mar. 13, 2016 Thawed in an
  								ice water bath and the
  								cells are very distorted.
  								Less than 1% motility and
  								a Live 9/91 Dead stain.
  101	May 3,	Apr. 29,	Odin	None	20 ul of	Unknown		Mar. 2, 2016 Thawed in an
  	2015,	2015			Semen	as		ice water bath at 41 F.
  	Mar. 2,				plus 60 ul	sample		Yellow straw. Cells very
  	2016				Purdy 10%,	from		deformed. Live 3/97 Dead
  	Yellow				Maple sap	holder #5		stain.
  	straw,					was lost
  	Holder #6					from
  						straw.,
  						Maybe
  102	May 10,	Apr. 30,	Odin	None	52 ul of	Maybe,		Processed at room
  	2015,	2015			Semen	Maybe,		temperature, placed
  	Mar. 2,				plus 104 ul	Maybe-		between 43 F. gel packs,
  	2016				of Purdy	Many		for 2 minutes and then
  	Orange				5% Maple	swollen		flash frozen.
  	straw from				Sap	and		May 10, 2015 Thawed in 70 F.
  	Holder #6,					distorted		water bath, Live dead
  	Mar. 6,					cells.		stain; 28 live/72 dead,
  	2016							Mar. 2, 2016 Orange soda
  	Orange							straw. Thawed in an ice
  	straw from							water bath at 41 F. Had
  	Holder #6.							10% forward motility with
  								many deformed cells. The
  								speed of motility increased
  								with warming. It had a Live
  								33/67 Dead stain.
  								Mar. 6, 2016 Thawed in an
  								ice water bath at 41 F. It
  								had 10-15% forward
  								motility and many distorted
  								and swollen cells. Live
  								21/79 Dead. Motility speed
  								increased with warming.
  103	May 3,	Apr. 30,	Odin	None	65 ul of	NO,		No acclimation in the
  	2015,	2015			Semen,	maybe,		fridge. Flash frozen.
  	May 10,				plus Purdy	Yes =		Thawed May 3, 2015 in Ice
  	2016,				10% Maple	Quiescent,		water, pH 7.5, 0% motility,
  	Mar. 21,				Sap, plus	Yes =		no live dead stain done,
  	2016				5% (13 ul)	Quiescent		Sample thawed May 10, 2015
  	Green				of DMA, No			Ice water bath, pH 7, 2-3%
  	straw				acclimation			motility, Live Dead Stain
  	Holder #6,							Live 23/Dead 77.
  	Mar. 13,							Mar. 2, 2016 Thawed in an
  	2016							ice water bath at 41 F.
  	Green							Saw 2% motile Almost no
  	straw							gross cell deformity with a
  	Holder #6							Live 52/48 Dead stain.
  								The longer it warmed on
  								the plate the higher the
  								motility up to 4%.
  								Mar. 13, 2016 Thawed in an
  								ice water bath at 41 F.
  								Less than 1% motility.
  								Little cell distortion and a
  								Live 20/80 Dead stain.
  104	May 3,	May 1,	Odin	None	8 ul of	No		May 3, 2015 Cells do not do
  	2015	2015			Semen			well with this at room
  					plus 8 ul			temperature. Thawed in
  					Purdy 10%			ice water, less than 2%
  					Maple tree			motile, No live/dead stain
  					sap plus			done.
  					Arabogalactin,
  					(No
  					MA), Plus
  					Purdy 10%
  					Maple Sap
  					plus
  					Arabogalactin
  					plus
  					12% MA
  105	Apr. 4,	Mar. 25,	Odin	None	55 ul of	Maybe;		Apr. 4, 2016 Thawed in a ice
  	2016,	2016			Semen;	Mistake		water bath. No motility and
  	Apr. 8,				plus 55 ul	made		has a Live 54/46 Dead
  	2016,				of BTE	adjusting		stain.
  	Apr. 10,				with/out	pH up in		Apr. 8, 2016 Thawed in an
  	2016				fructose	2nd		ice water bath at 41 F. and
  					plus ½%	media		then palmed to warm. No
  					Sucrose;	used -		motility with a Live 4/96
  					plus 55 ul	using		Dead stain.
  					of BTE w/o	Bicarb		Apr. 10, 2016 Thawed in an
  					Fruc + 1st	changing		ice water bath at 41 F. No
  					Run maple	the pH		motility with a Live 12/88
  					tree sap,	from 6.48		Dead stain.
  					Tree #3.	to 7.23.
  					2015	This
  						appears
  						to impair
  						motility.
  						(Bad),
  						Used 30
  						drops of
  						Bicarb
  						from a
  						low dose
  						insulin
  						syringe to
  						a 10 cc
  						tube.
  106	Apr. 3,	Mar. 26,		None	50 ul of	Maybe;		Apr. 3, 2016 Acellular, pH of
  	2016,	2016			Semen; 50	Mistake		8 on tape, No cells seen
  	Apr. 8,				ul of BTE −	made		on L/D stain.
  	2016,				Fruc +	adjusting		Apr. 8, 2015 Ice water thaw
  	Apr. 10,				½% Suc;	pH up in		and then palmed to warm.
  	2016				Plus 100 ul	2nd		pH near 8 on tape. No
  					of BTE −	media		cells seen, lysed. No L/D
  					Fruc + 1st	used -		stain.
  					Run Maple	using		Apr. 10, 2016 Ice water thaw,
  					tree sap.	Bicarb		pH near 7.5 on tape. No
  					2015	changing		motility seen. Live 21/79
  						the pH		Dead.
  						from 6.48
  						to 7.23.
  						This
  						appears
  						to impair
  						motility.
  						(Bad),
  						Used 30
  						drops of
  						Bicarb
  						from a
  						low dose
  						insulin
  						syringe to
  						a 10 cc
  						tube.
  107	Apr. 8,	Mar. 26,	Odin	None	47 ul of	Maybe;		Apr. 4, 2016 Ice water thaw,
  	2016,	2016			Semen;	Mistake		pH 7 on tape. No motility.
  	Apr. 4,				Plus 23 ul	made		Live 53/47 Dead stain.
  	2016,				of BTE −	adjusting		Apr. 8, 2016 Ice water thaw
  	Apr. 8,				Fruc + ½%	pH up in		and then palmed to warm.
  	2016				Suc;	2nd		pH on tape was 7.2. No
  					Plus 65 ul	media		motility. Live 15/85 Dead
  					of BTE −	used -		stain.
  					Fruc + 1st	using		Apr. 8, 2016 Ice water thaw
  					run Maple	Bicarb		and then palmed to warm.
  					Tree sap.	changing		pH 7.5 on tape. Live 13/87
  					2015	the pH		Dead.
  						from 6.48
  						to 7.23.
  						This
  						appears
  						to impair
  						motility.
  						(Bad).
  						Used 30
  						drops of
  						Bicarb
  						from a
  						low dose
  						insulin
  						syringe to
  						a 10 cc
  						tube.
  108	Apr. 4,	Mar. 27,	Odin	None	40 ul	Maybe;		Apr. 4, 2016 Capillary tube
  	2016,	2016			Semen;	Mistake		lost from straw in the tank.
  	Apr. 10,				Plus 20 ul	made		Apr. 10, 2016 Ice water thaw.
  	2016,				of BTE −	adjusting		No motility. pH of 7.2. No
  	Apr. 10,				Fruc +	pH up in		L/D stain.
  	2016				½% Suc;	2nd		Apr. 10, 2016 Ice water thaw.
  					Plus 40 ul	media		pH 7.2 on tape. Less than
  					of BTE −	used -		1% motile. Live 24/76
  					Fruc + 1st	using		Dead.
  					Run Maple	Bicarb
  					Tree #3.	changing
  					2015	the pH
  						from 6.48
  						to 7.23.
  						This
  						appears
  						to impair
  						motility.
  						(Bad).
  						Used 30
  						drops of
  						Bicarb
  						from a
  						low dose
  						insulin
  						syringe to
  						a 10 cc
  						tube.
  109	Apr. 4,	Mar. 27,	Odin	None	40 ul of	Maybe;		Apr. 4, 2016 Ice water thaw,
  	2016,	2016			Semen;	Mistake		pH 8 on tape. No motility
  	Apr. 4,				Plus 40 ul	made		and a Live 30/70 Dead
  	2016,				of BTE −	adjusting		stain.
  	Apr. 10,				Fruc +	pH up in		Apr. 4, 2016 Ice water thaw.
  	2016,				½% Suc;	2nd		pH on tape of 7.2. No
  	Apr. 10,				Plus 80 ul	media		motility. Live 30/70 Dead
  	2016				of BTE −	used -		stain.
  					Fruc + 1st	using		Apr. 10, 2016 Ice water thaw
  					Run Maple	Bicarb		with pH of 7.5 on tape. No
  					Tree #3.	changing		motility and a Live 19/81
  					2015	the pH		Dead stain.
  						from 6.48		Apr. 10, 2016 Ice water thaw
  						to 7.23.		pH of 7.5.
  						This
  						appears
  						to impair
  						motility,
  						(Bad).
  						Used 30
  						drops of
  						Bicarb
  						from a
  						low dose
  						insulin
  						syringe to
  						a 10 cc
  						tube.
  110	Apr. 3,	Mar. 28,	Odin	None	30 ul of	Maybe;		Apr. 3, 2016 Ice water thaw,
  	2016,	2016			Semen;	Mistake		pH on tape 7.2. No
  	Apr. 5,				Plus 30 ul	made		motility. Live 51/49 Dead
  	2016,				of BTE −	adjusting		stain.
  	Apr. 10,				Fruc + ½	pH up in		Apr. 5, 2016 Ice water thaw,
  	2010				% Suc;	2nd		pH of 7 on tape. No
  					Plus BTE −	media		motility. Live 5/95 dead
  					Fruc + 1st	used -		stain.
  					Run Maple	using		Apr. 10, 2016 Ice water thaw.
  					Tree sap.	Bicarb		pH of 7.2 on tape. No
  					2015	changing		motility. Live 16/84 Dead
  						the pH		stain.
  						from 6.48
  						to 7.23.
  						This
  						appears
  						to impair
  						motility.
  						(Bad).
  						Used 30
  						drops of
  						Bicarb
  						from a
  						low dose
  						insulin
  						syringe to
  						a 10 cc
  						tube.
  111	Apr. 4,	Mar. 28,	Odin	None	40 ul of	Maybe;		Apr. 4, 2016 Ice water thaw,
  	2016,	2016			Semen;	Mistake		pH of 7.2 on tape. No
  	Apr. 8,				Plus 40 ul	made		motility and a Live 35/65
  	2016,				of BTE −	adjusting		Dead stain.
  	Apr. 8,				Fruc +	pH up in		Apr. 8, 2016 Ice water thaw
  	2016				½% Suc;	2nd		and then palmed to warm.
  					Plus 60 ul	media		pH of 7.2 on tape. Live
  					of BTE −	used -		5/95 Dead.
  					Fruc + 1st	using		Apr. 8, 2016 Less than 1%
  					Run Maple	Bicarb		motility on ice water thaw.
  					Tree #3.	changing		pH 7.2 on tape. Live 7/93
  					2015	the pH		Dead.
  						from 6.48
  						to 7.23.
  						This
  						appears
  						to impair
  						motility.
  						(Bad).
  						Used 30
  						drops of
  						Bicarb
  						from a
  						low dose
  						insulin
  						syringe to
  						a 10 cc
  						tube.
  112	Apr. 4,	Mar. 30,	Odin	None	42 ul of	Yes, one		Apr. 4, 2016 Ice water thaw.
  	2016,	2016			Semen;	sample		pH of 7.5 on tape. Live
  	Apr. 5,				Plus 42 ul	did very		25/75 Dead stain.
  	2016,				of BTE, no	well and I		Apr. 5, 2015 Ice water thaw
  	Apr. 5,				fructose +	wonder if		and then palmed, 50%
  	2016				½% Suc	it froze		motile, pH 7.5 on pH tape,
  					(pH 7.4);	more		Live 53/47 Dead stain.
  					Plus 65 ul	slowly.		Apr. 5, 2016 Ice water thaw
  					of BTE −			and then palmed to warm.
  					Fruc + plus			No motility. Live 8/92
  					1st Run			Dead. Need slow freeze
  					Maple Tree			and at least 30% sap for
  					Sap from			the cells to survive. pH
  					Tree #3			adjustments bad on
  					(2015), (pH			these samples.
  					7.23)
  113	Apr. 4,	Mar. 30,	Odin	None	35 ul of	Maybe;		Apr. 4, 2016 Ice water thaw,
  	2016,	2016			Semen;	pH		no cells seen. No pH
  	Apr. 5,				Plus 35 ul	adjustment		done. No L/D stain.
  	2016,				of BTE −	in both		Apr. 5, 2016 Ice water thaw.
  	Apr. 8,				Fruc + ½%	diluents is		No motility. No pH done.
  	2016				Suc (pH	stressing		Live 19/81 Dead stain.
  					7.4); Plus	the cells		Apr. 8, 2016 Ice water thaw.
  					35 ul of	too much		pH 7.3. No motility. Live
  					BTE − Fruc +	and stops		12/88 Dead stain.
  					1st run	motility.
  					Maple Tree
  					sap tree
  					#3. 2015.
  					(pH 7.23).
  114	Apr. 4,	Apr. 2,	Odin	None	25 ul of	Maybe;		Apr. 4, 2016 Ice water thaw
  	2016,	2016			Semen;	the pH		with no motility. pH 8 on
  	Apr. 5,				Plus 25 ul	and		tape. Live 26/73 Dead
  	2016,				BTE − Fruc +	speed of		stain.
  					½% Suc	freezing		Apr. 5, 2016 Ice water thaw
  					(pH 7.4);	need to		and no motility. pH of 7.4
  					Plus 35 ul	be		on tape. Live 10/90 Dead
  					of BTE −	changed.		stain. Too little sap was
  					Fruc + 1st	Slow the		used and the freezing
  					Run Maple	freeze		needs to be slow. pH
  					tree sap	down and		needs to be lower in
  					(pH 7.23).	lower the		starting extenders.
  					2015. No	pH to
  					other	decrease
  					cryoprotectant.	cell
  						metabolism.
  115	Apr. 3,	Apr. 3,	Odin	None	65 ul	Maybe;		Apr. 3, 2016 Ice water thaw,
  	2016,	2016			Semen;	the pH		pH on tape 7.4, No motility
  	Apr. 5,				Plus 65 ul	and		and a Live 17/84 Dead
  	2016,				of BTE −	speed of		stain. Percentage of sap
  	Apr. 5,				Fruc = ½%	freezing		needs to increase from
  	2016,				Suc (pH	need to		33%.
  	Apr. 5,				7.4); Plus	be		Apr. 5, 2016 lost sample as
  	2016				65 ul BTE −	changed.		the caulk came out on
  					Fruc + 1st	Slow the		thawing.
  					Run Maple	freeze		Apr. 5, 2016 Ice water thaw
  					Tree sap.	down and		and then palmed to warm
  					2015 (pH	lower the		because caulk came out.
  					7.23)	pH to		pH 7.5 on tape. No
  						decrease		motility. Live 8/92 Dead
  						cell		stain.
  						metabolism.		Apr. 5, 2016 Ice water thaw
  								and then palmed to warm.
  								pH 7.2. No motility. Live
  								6/94 Dead.
  116	Apr. 4,	Apr. 4,	Odin	None	40 ul of	NO, NO,		Apr. 4, 2016 Ice water thaw.
  	2016,	2016			Semen;	NO,		pH of 7. Live 0/100 Dead
  	Apr. 4,				Plus 40 ul	Formula		stain. No motility.
  	2016,				of	error		Apr. 4, 2016 Ice water thaw.
  	Apr. 4,				Goshawk	stopping		pH of 7. Live 25/75 Dead
  	2016				Semen	motility in		stain. No motility.
  					Extender =	samples.		Apr. 4, 2016 Ice water thaw.
  					BTE − Fruc +	The		pH of 7. Live 9/91 Dead
  					½%	addition		stain. No motility.
  					Suc w/pH	of
  					of 6.27.:	glutathione
  					Plus 80 ul	and
  					BTE − Fruc +	NN-
  					Amur	Bis . . . sulfonic
  					Maple +	acid
  					Glutathione +	should
  					NN-	not have
  					Bis . . . Sulfonic	been
  					Acid	done.
  117	Apr. 4,	Apr. 4,	Odin	None	40 ul	NO, NO,		Apr. 4, 2016, Ice water thaw,
  	2016,	2016			Semen;	NO,		pH 7, No motility. Live
  	Apr. 4,				Plus 40 ul	Formula		28/72 Dead.
  	2016,				Goshawks	error		Apr. 4, 2016 Ice water thaw.
  	Apr. 4,				Extender	stopping		pH 7. No motility. Live
  	2016				(pH 6.27);	motility in		0/100 Dead stain.
  					Plus 80 ul	samples.		Apr. 4, 2016 Ice water thaw.
  					of BTE −	The		pH 7, No motility. Sample
  					Fruc +	addition		too small for a Live/Dead
  					Amur	of		stain. 1 straw missing.
  					Maple 1st	glutathione
  					run. +	and
  					Glutathione +	NN-
  					NN	Bis . . . sulfonic
  					Bis . . . sulfonic	acid
  					acid.	should
  						not have
  						been
  						done.
  118	Apr. 5,	Apr. 5,	Odin	None	33 ul	Yes,	Brix value	Apr. 5, 2016 Ice water thaw,
  	2016, ( )	2016			Semen;	simple	2.5	pH on tape was 7, 15-20%
  					Plus 66 ul	addition		moving initially. Live
  					BTE −	of sap		47/Dead 53. Longer
  					Fructose +	allowed		acclimation may have
  					Maple tree	the cells		increased survival. But
  					sap tree #3	to		longer acclimation
  					2015. Did	survive.		decreases survival in the
  					not			hen's seminal tubules
  					acclimate			when it does not have an
  					with an			extender added.
  					extender.
  					Only
  					acclimated
  					semen in
  					its own
  					tube and
  					then added
  					the sap.
  119	Apr. 10,	Apr. 5,	Odin		Back to the	Yes, Yes	Brix value	Apr. 10, 2016, Ice water thaw,
  	2016,	2016			2015		2.5	pH 7 on test tape, About
  	Apr. 11,				Formulas.			5% vibrating in place, Live
  	2016, ( ).				The			30/Dead 70. Would likely
  					modification			have done better if left
  					that I			above the vapors longer.
  					made to			Apr. 11, 2016 Ice water thaw,
  					the pH was			pH 7 on tape, Some
  					stopping			vibrating in place but not
  					motility in			moving forward, Live
  					the cells so			31/Dead 69. It takes 90
  					I started			seconds to process 1
  					back at			sample into 4 tubes.
  					baseline
  					extenders.
  					45 ul
  					Semen;
  					Plus 45 ul
  					of BTE −
  					Fructose +
  					½%
  					Sucrose
  					(pH 7.51);
  					Plus 90 ul
  					of BTE −
  					Fructose +
  					Maple Tree
  					#3 2015
  					(pH 6.48)
  120	Apr. 10,	Apr. 8,	Odin		Back to the	Yes, the		Apr. 10, 2016 Ice water thaw,
  	2016	2016			2015	original		pH 7, 50% initially motile
  					Formulas.	2015 sap		to about 5% motile over
  					The	formulas		about 10 minutes. Live
  					modification	support		62/Dead 38. It took 90
  					that I	the cells		seconds to package the 4
  					made to	well.		straws and this delay in
  					the pH was			getting it into the LN2
  					stopping			likely allowed the osmotic
  					motility in			gradient across the cells to
  					the cells so			fade, allowing the cell to
  					I started			rehydrate prior to the
  					back at			freeze. This is conjecture,
  					baseline			but noted as a problem in
  					extenders.			references. Need a
  					45 ul			processing time less than
  					Semen,			this. I stored a small
  					then 45 ul			sample of this tube in the
  					of BTE −			fridge from 7:30 AM to
  					Fructose +			1:30 AM and more than
  					½%			75% were moving forward
  					Sucrose			and straight. (not frozen).
  					(pH 7.51),
  					then added
  					90 ul of
  					BTE −
  					Fructose +
  					Maple Tree
  					#3 2015 (p
  121		Apr. 6,	Odin		Back to the		Brix
  		2016			2015		value
  					Formulas.		2.5
  					The
  					modification
  					that I
  					made to
  					the pH was
  					stopping
  					motility in
  					the cells so
  					I started
  					back at
  					baseline
  					extenders.
  					55 ul
  					Semen;
  					Plus 55 ul
  					of BTE −
  					Fructose +
  					½%
  					Sucrose
  					(pH 7.51);
  					Plus 100 ul
  					of BTE −
  					Fructose +
  					Maple Tree
  					#3 2015
  					(pH 6.48)
  122		Apr. 7,	Odin		Back to the		Brix
  		2016			2015		value
  					Formulas.		2.5
  					The
  					modification
  					that I
  					made to
  					the pH was
  					stopping
  					motility in
  					the cells so
  					I started
  					back at
  					baseline
  					extenders.
  123		Apr. 8,	Odin		Back to the		Brix
  		2016			2015		value
  					Formulas.		2.5
  					The
  					modification
  					that I
  					made to
  					the pH was
  					stopping
  					motility in
  					the cells so
  					I started
  					back at
  					baseline
  					extenders.
  124		Apr. 7,	Odin		Back to the		Brix
  		2015			2015		value
  					Formulas.		2.5
  					The
  					modification
  					that I
  					made to
  					the pH was
  					stopping
  					motility in
  					the cells so
  					I started
  					back at
  					baseline
  					extenders.
  125		Apr. 8,	Odin		Back to the		Brix
  		2016			2015		value
  					Formulas.		2.5
  					The
  					modification
  					that I
  					made to
  					the pH was
  					stopping
  					motility in
  					the cells so
  					I started
  					back at
  					baseline
  					extenders.
  126	Apr. 10,	Apr. 8,	Odin		Back to the	Maybe,	Brix	Holder #4 trace sample
  	2016,	2016			2015	YES	value	because caulk came out of
  	Apr. 11,				Formulas.		2.5	capillary tube, Thawed in
  	2016				The			Ice Water, No pH, No L/D
  					modification			stain. 10% moving forward
  					that I			with good motility.;
  					made to			Apr. 11, 2016 Ice water thaw,
  					the pH was			pH 7, 30% motile on visual
  					stopping			inspection, Live 50/Dead
  					motility in			50.
  					the cells so
  					I started
  					back at
  					baseline
  					extenders.
  					40 ul
  					Semen;
  					Plus 40 ul
  					of BTE −
  					Fructose +
  					½%
  					Sucrose
  					2015 (pH
  					7.51); Plus
  					80 ul BTE −
  					Fructose +
  					Maple Tree
  					sap #3
  					2015
  					(pH 6.48).
  127		Apr. 10,	Odin		Back to the
  		2016			2015
  					Formulas.
  					The
  					modifications
  					that I
  					made to
  					the pH was
  					stopping
  					motility in
  					the cells so
  					I started
  					back at the
  					baseline
  					extenders.
  					BIRCH sap
  					STARTS
  					HERE.
  					40 ul
  					Semen,
  					plus 40 ul
  					of BTE −
  					Fructose +
  					1st Run
  					Alaska
  					Birch (pH.
  					7.56), then
  					added 40 ul
  					BTE −
  					Fructose +
  					Maple Tree
  					#3 2015
  					(pH. 6.48).
  128	Apr. 10,	Apr. 10,	Odin		40 ul	Yes		Apr. 10, 2016 Ice water thaw,
  	2016	2016			Semen;			pH 7 on tape, 25% moving
  					Plus 40 ul			straight forward, Live
  					BTE −			57/43 Dead stain.
  					Fructose +
  					1st run
  					Alaska
  					Birch
  					(pH. 7.56);
  					Plus 40 ul
  					BTE −
  					Fructose +
  					Maple Tree
  					sap tree #3
  					2015
  					(pH 6.48).

  		How sample was			Holder in tank it		How storage
  	Number	frozen.	pH of Diluent	Diluent Type	is located in	Straw Type.	straw performed.
  	4	Either flash frozen or	9.1	Diluent #1	Unknown.	Unknown
  		suspended above the
  		liquid nitrogen for 30
  		seconds prior to
  		immersion.
  	5	Flash frozen.	9.1	Diluent #1	2	Unknown
  	6	Either flash frozen or	9.1	Diluent #1	Unknown.	Natellson	This straw
  		suspended above the				Capillary	for storage
  		liquid nitrogen for 30				tube with 2	stored well
  		seconds prior to				caps.	and did not
  		immersion.					explode.
  	7	Either flash frozen or	9.1	Diluent #1	Unknown.	Unknown
  		suspended above the
  		liquid nitrogen for 30
  		seconds prior to
  		immersion.
  	8	Flash frozen.	8.86	Diluent #2	2	Unknown
  	9	Either flash frozen or	8.86	Diluent #2	Unknown.	Unknown
  		suspended above the
  		liquid nitrogen for 30
  		seconds prior to
  		immersion.
  	10	Flash frozen.	8.86	Diluent #2	2
  	11	Either flash frozen or	8.86	Diluent #2	Unknown.	Unknown
  		suspended above the
  		liquid nitrogen for 30
  		seconds prior to
  		immersion.
  	12	Flash frozen.	8.86	Diluent #2	2	Unknown
  	13	Either flash frozen or	8.86	Diluent #2	Unknown.	Unknown
  		suspended above the
  		liquid nitrogen for 30
  		seconds prior to
  		immersion.
  	14	Either flash frozen or	9.08	Diluent #3	Unknown.	Unknown
  		suspended above the
  		liquid nitrogen for 30
  		seconds prior to
  		immersion.
  	15	Either flash frozen or	Unknown	None	Unknown.	Unknown
  		suspended above the
  		liquid nitrogen for 30
  		seconds prior to
  		immersion.
  	16	Either flash frozen or	9.08	Diluent #3	Unknown.	Unknown
  		suspended above the
  		liquid nitrogen for 30
  		seconds prior to
  		immersion.
  	17	Flash frozen.	9.08	Diluent #3	3	Natellson	This straw
  						Capillary	for storage
  						tube with 2	stored well
  						caps.	and did not
  							explode.
  	18	Flash frozen.	9.18	Diluent #4	2	Natellson
  						Capillary
  						tube with 2
  						caps.
  	19	Either flash frozen or	7.06	Trout	Unknown.	Natellson
  		suspended above the				Capillary
  		liquid nitrogen for 30				tube with 2
  		seconds prior to				caps.
  		immersion.
  	20	Either flash frozen or	7.06	Trout	Unknown.	Natellson	Exploded
  		suspended above the				Capillary	and lost.
  		liquid nitrogen for 30				tube with 2
  		seconds prior to				caps.
  		immersion.
  	21	Flash frozen.	7.06	Trout	2	Natellson
  						Capillary
  						tube with 2
  						caps.
  	22	Flash frozen.	7.06	Trout	5	Unknown
  	23	Suspended over liquid	7.16	Trout #2	Unknown.	Natellson	This straw
  		nitrogen for 30 seconds				Capillary	for storage
  		to exposed to liquid				tube with 2	stored well
  		nitrogen vapors and then				caps.	and did not
  		flash frozen.					explode.
  	24	Suspended over liquid	7.16	Trout #2	Floating	Natellson	Exploded
  		nitrogen for 30 seconds			on liquid	Capillary	on thaw
  		to exposed to liquid			nitrogen	tube with 2	but did not
  		nitrogen vapors and then			and lost	caps.	lose the
  		flash frozen.			out of		sample.
  					holder.
  	25	Flash frozen.	7.16	Trout #2	5	Unknown
  	26	Flash frozen.	7.16	Trout #2	5	Unknown
  	27	Flash frozen.		None	5	Unknown
  	28	Flash frozen.	7.16	Trout #2	5	Unknown
  	29	Suspended over liquid	7.14	Milk #1	Unknown.	Natellson	This straw
  		nitrogen for 30 seconds				Capillary	for storage
  		to exposed to liquid				tube with 2	stored well
  		nitrogen vapors and then				caps.	and did not
  		flash frozen.					explode.
  	30	Flash frozen.	7.42	Trout #2	5	Unknown
  				plus
  				Sorbitol
  				and
  				Arabogalactin.
  	31	10 mm of semen to 45		Turkey	6	Commercial	Exploded
  		mm of Turkey Extender,		Extender		semen	and lost.
  		Acclimated in the fridge		plus DMA		straw with
  		for 30 minutes, Added 3				button
  		ul (units) of DMA, Hung				caps.
  		in vapors for 10 minutes				GREEN
  		the placed in liquid				ON TOP
  		nitrogen.				OF
  						ALUMINUM
  						HOLDER.
  	32	Semen acclimated in		Turkey	6	Commercial	FOUND
  		fridge for 30 minutes,		Extender		semen	FLOATING
  		then DMA added, then		plus (6%)		straw with	IN TANK
  		hung over vapors for 10		DMA		button	MINUS
  		minutes, then immersed				caps.	LABEL
  		in liquid nitrogen.				GREEN
  						ON TOP
  						OF
  						ALUMINUM
  						HOLDER.
  	33	Semen acclimated in		Turkey	6	Commercial	FOUND
  		fridge for 30 minutes,		Extender		semen	FLOATING
  		then DMA added, then		plus DMA		straw with	IN TANK
  		hung over vapors for 10				button	MINUS
  		minutes, then immersed				caps.	LABEL
  		in liquid nitrogen.				GREEN
  						ON TOP
  						OF
  						ALUMINUM
  						HOLDER.
  	34	Semen acclimated in		Turkey	6	Commercial	FOUND
  		fridge for 30 minutes,		Extender		semen	FLOATING
  		then DMA added, then		plus DMA		straw with	IN TANK
  		hung over vapors for 10				button	MINUS
  		minutes, then immersed				caps.	LABEL
  		in liquid nitrogen.				GREEN
  						ON TOP
  						OF
  						ALUMINUM
  						HOLDER.
  	35	Semen acclimated in		Turkey	6	Commercial	Exploded
  		fridge for 30 minutes,		Extender		semen	and not
  		then DMA added, then		plus DMA		straw with	lost. But
  		hung over vapors for 10				button	explosion
  		minutes, then immersed				caps.	was loud
  		in liquid nitrogen.				GREEN	and semen
  						ON TOP	cells likely
  						OF	damaged
  						ALUMINUM	due to
  						HOLDER.	trauma. I
  							am
  							guessing
  							that this
  							sample
  							went to
  							Juniper
  							because 4
  							were found
  							floating
  							free on top
  							of the liquid
  							nitrogen
  							and not
  							labeled.
  	36	See prior note		Turkey	6	Unknown
  				Extender
  				plus DMA
  	37	Flash frozen.		Turkey	5	Commercial	Exploded
  				Extender		semen	and lost.
  				plus DMA		straw with
  						button
  						caps. RED
  						ON TOP
  						OF
  						ALUMINUM
  						HOLDER
  	38	Flash frozen.		Turkey	5	Commercial	Exploded
  				Extender		semen	and lost.
  				plus		straw with
  				(10%)		button
  				DMA		caps. RED
  						ON TOP
  						OF
  						ALUMINUM
  						HOLDER
  	39	Flash frozen.		Turkey	5	Commercial	Exploded
  				Extender		semen	and lost.
  				plus		straw with
  				(10%)		button
  				DMA		caps. RED
  						ON TOP
  						OF
  						ALUMINUM
  						HOLDER
  	40	Flash frozen.		Turkey	5	Commercial	Exploded
  				Extender		semen	and lost.
  				plus		straw with	The
  				(18%)		button	reinforced
  				DMA		caps. RED	plastic
  						ON TOP	coated
  						OF	glass
  						ALUMINUM	capillary
  						HOLDER	tubes
  							withstand
  							explosions.
  	41	SEE NOTES ON 2013		Turkey
  		SAMPLES.		Extender
  				plus DMA
  	42	In fridge 10 minutes to	6.74	Turkey	#2	#42, white
  		acclimate, plus 1.5 units		Extender		straw,
  		DMA, above vapors for		plus DMA,		Natellson
  		10 minutes, immersed		5% DMA		tube,
  		suddenly into LN2				capped on
  						1 end firmly
  						and outside
  						end not
  						capped.
  	43	Acclimated in fridge 10	6.74	Turkey	#2	#43 White
  		minutes, plus 1.5 units		Extender		straw,
  		DMA (5%), hung over		plus DMA,		standard
  		vapors for 10 minutes		5% DMA		capillary
  		within 1 minute of adding				tube, plus
  		DMA, dunked into LN2.				two crito
  						caps and 1
  						Natellson
  						cap.
  	44	Acclimated in fridge 10	6.74	Turkey	#2	#44 White	Straw
  		minutes, plus 3 ul of		Extender		straw,	exploded
  		DMA (6%), Acclimated in		plus 6%		standard	on thaw,
  		fridge for 10 minutes,		DMA		capillary	but one
  		suspended above the				tube plus 2	end stayed
  		vapors 10 minutes, then				critocaps	closed.
  		dunked in LN2.				and 1
  						Natellson
  						cap.
  	45	Acclimated in fridge 10	6.74	Turkey	#2	#45 White
  		minutes, plus 2 ul of		extender		straw,
  		DMA (10%), hung over		plus 10%		Standard
  		vapors for 10 minutes,		DMA		capillary
  		plunged into LN2				tube, plus 2
  						caps on
  						one end
  						and clay on
  						the far end.
  	46	Acclimated in fridge (35	6.74	Turkey	#2	#46 White
  		F.), Plus 2 ul of DMA,		extender		straw, tube
  		hung over vapors for 10		plus 10%		type not
  		minutes, plunged into		DMA		recorded.
  		LN2.
  	47	Acclimated in fridge at 35	6.74	Turkey	#2	#47, two
  		F. for 10 minutes, one		Extender		tubes,
  		sample hung over vapors		plus 6%		White
  		for 7 minutes and the		DMA		straw,
  		other 10 minutes, and				Natellson
  		then dunked into LN2.				tube, plus
  						clay and
  						Natellson
  						cap on
  						pointed
  						end, and
  						Natellson
  						cap on top
  						end.
  	48	Acclimated in the fridge	6.74	Turkey	#2	White
  		at 35 F., 1.5 ul of DMA		extender		straw,
  		added, over vapors 10		plus 7%		Natellson
  		minutes, dunked into		DMA.		capillary
  		LN2.				tube plus
  						clay in both
  						ends and
  						Natellson
  						cap on
  						pointed
  						end.
  	49	Acclimated in fridge at	6.74	Turkey	#2	White
  		35 F. for 10 minutes, plus		Extender		straw,
  		3 ul of DMA, hung over		plus 6%		Natellson
  		vapors 10 minutes,		DMA		tube plus
  		plunged into LN2.				clay on
  						both ends
  						and rubber
  						cap on
  						pointed
  						end.
  	50	Acclimated in fridge at	6.74	Turkey	#2	White
  		35 F. for 10 minutes, plus		extender		straw, 75 ul
  		2 ul of DMA (7%), over		plus 7%		standard
  		vapors for 10 minutes,		DMA.		capillary
  		then plunges into LN2.				tube plus 1
  						critocap
  						and
  						Natellson
  						Cap on one
  						end and
  						Clay on the
  						other end.
  	51	Acclimated in the fridge	6.74	Turkey	#2	White
  		at 35 F., 1.25 (5%) DMA		extender		straw, 75 ul
  		added, hung over vapors		with 5%		standard
  		for 10 minutes, plunged		DMA.		capillary
  		into LN2.				tube plus
  						one end a
  						critocap
  						and one
  						end a
  						Natellson
  						cap.
  	52	Acclimated in the fridge	6.74	Turkey	#2	White
  		at 35 F., 2.7 ul of DMA		extender		straw,
  		added, Hung over vapors		with 6%		Natellson
  		10 minutes, plunged into		DMA.		Capillary
  		LN2.				tube, plus
  						clay on
  						both ends,
  						and
  						Natellson
  						cap on
  						pointed
  						end.
  	53	Acclimated in the fridge	6.74	Turkey	#2	White
  		at 35 F. for 15 minutes,		extender		straw,
  		2.23 ul of DMA added,		with 6%		Natellson
  		hung over vapors 10		DMA.		Capillary
  		minutes, plunged into				tube, plus
  		LN2.				clay on
  						both ends,
  						and
  						Natellson
  						cap on
  						pointed
  						end.
  	54	Acclimated in the fridge	6.74	Turkey	#2	White
  		at 35 F. for 15 minutes,		extender		straw,
  		plus 1 ul of DMA (5%),		with 6%		Standard
  		hung over vapors 10		DMA.		75 ul
  		minutes, plunged into				capillary
  		LN2.				tube, clay
  						on one end
  						and
  						Natellson
  						cap and
  						critocap on
  						other end.
  	55	Acclimated in the fridge	6.74	Turkey	#2	White
  		at 35 F. for 15 minutes,		extender		straw,
  		added 2.5 ul DMA, hung		with 5%		Natellson
  		over vapors 10 minutes,		DMA.		tube plus
  		plunged into LN2.				clay on
  						pointed end
  						and
  						Natellson
  						caps on
  						both ends.
  	56	Acclimated in the fridge	6.74	Turkey	#2	Red Straw,
  		for 15 minutes, plus 1 ul		extender,		Natellson
  		of DMA (5%), hung over		with		tube, with
  		vapors for 10 minutes,		Maple		clay and
  		plunged into LN2.		Syrup,		Natellson
  				with 5%		cap on
  				DMA.		pointed end
  						and just
  						clay on
  						large end.
  	57	Acclimated in the fridge		Turkey	#4 holder	Red straw.	The caps
  		for 10 minutes, plus 1 ul		Extender	in Tank 2	This was a	exploded
  		of DMA (5%), dropped		by itself		75 ul mylar	off of the
  		into the LN2.				coated	ends and
  						capillary	most of the
  						tube,	sample
  						caulked on	was lost.
  						both ends,
  						plus 2
  						critocaps,
  						plus a teal
  						cap on the
  						semen end.
  	58	Acclimated in the fridge		Beltsville	#4 Holder	Red soda	Sample
  		for 15 minutes, plus 3 ul		Turkey	in Second	straw, with	explodes
  		of DMA		Extender	tank.	a 75 ul	across the
  				plus .2 mg		Mylar	garage.
  				Inositol to		coated
  				10 ml of		capillary
  				Extender.		tube,
  						caulked on
  						both ends,
  						with two
  						critocaps,
  						and one
  						teal cap.
  						Plus
  						aluminum
  						holder.
  	59	Acclimated in the fridge		Beltsville	#4 Holder	Red soda	Tube
  		for 15 minutes, Plus 2.6 ul		Turkey	in the	straw, with	performed
  		of DMA and then flash		Extender	Second	a 75 ul	well.
  		frozen.		plus .2 mg	tank	Mylar
  				Inositol to		coated
  				10 ml of		capillary
  				Extender.		tube,
  						caulked on
  						both ends,
  						with two
  						critocaps,
  						and one
  						teal cap.
  						With
  						Aluminum
  						holder.
  	60	Acclimated in the fridge		Beltsville	#4 Holder	Red soda	Exploded
  		for 15 minutes and then		Turkey	in the	straw, with	but kept
  		flash frozen		Extender	Second	a 75 ul	sample.
  				plus .2 mg	tank	Mylar
  				Inositol to		coated
  				10 ml of		capillary
  				Extender.		tube,
  						caulked on
  						both ends,
  						with two
  						critocaps,
  						and one
  						teal cap,
  						With
  						Aluminum
  						holder.
  	61	Acclimated in the fridge		Beltsville	#3 Holder,	Red soda	Straw
  		for 15 minutes and then		Turkey	Tank 2	straw, with	performed
  		flash frozen		Extender		a 75 ul	well.
  				plus .2 mg		Mylar
  				Inositol to		coated
  				10 ml of		capillary
  				Extender.		tube,
  						caulked on
  						both ends,
  						with two
  						critocaps,
  						and one
  						teal cap,
  	62	Acclimated in the fridge		Beltsville	#3 Holder,	Red soda	Straw
  		for 15 minutes and then		Turkey	Tank 2	straw, with	exploded
  		flash frozen		Extender		a 75 ul	on thaw,
  				plus .2 mg		Mylar	but sample
  				Inositol to		coated	was
  				10 ml of		capillary	preserved.
  				Extender.		tube,
  						caulked on
  						both ends,
  						with two
  						critocaps,
  						and one
  						teal cap.
  	63	Acclimated in the fridge		Beltsville	#3 Holder,	White soda	Tube
  		for 15 minutes and then		Turkey	Tank 2	straw, with	performed
  		flash frozen		Extender		a Mylar	well.
  				plus .2 mg		coated
  				Inositol to		capillary
  				10 ml of		tube,
  				Extender.		caulked on
  						both ends,
  						with two
  						critocaps,
  						and one
  						teal cap.
  	64	Acclimated in the fridge		Beltsville	#3 Holder,	Red soda	Tube
  		for 15 minutes and then		Turkey	Tank 2	straw, with	exploded,
  		flash frozen		Extender		a 75 ul	Lost most
  				plus .2 mg		Mylar	of the
  				Inositol to		coated	sample.
  				10 ml of		capillary
  				Extender.		tube,
  						caulked on
  						both ends,
  						with two
  						critocaps,
  						and one
  						teal cap.
  	65	Acclimated in the fridge		Beltsville	#3 Holder,	Red soda	Tube
  		for 15 minutes and then		Turkey	Tank 2	straw, with	performed
  		flash frozen		Extender		a 75 ul	well.
  				plus .2 mg		Mylar
  				Inositol to		coated
  				10 ml of		capillary
  				Extender.		tube,
  						caulked on
  						both ends,
  						with two
  						critocaps,
  						and one
  						teal cap.
  	66	Acclimated in the fridge		Beltsville	#3 Holder,	White soda	Straw
  		for 15 minutes and then		Turkey	Tank 2	straw, with	empty on
  		flash frozen		Extender		a Mylar	thaw.
  				plus .2 mg		coated
  				Inositol to		capillary
  				10 ml of		tube,
  				Extender.		caulked on
  						both ends,
  						with two
  						critocaps,
  						and one
  						teal cap,
  						No sample
  						in tube on
  						thaw.
  	67	No acclimation. Flash		SAP	#2 Holder	Orange	Trace
  		Frozen.		Beltsville	Tank 2	soda straw,	sample left
  				Turkey		with 75 ul	on thaw
  				Extender		capillary	due to
  				minus the		tube, plus	exploding.
  				fructose,		clay both
  				plus		ends, plus
  				Maple		critocaps.
  				Tree sap,
  				first run,
  				Tree
  				number 3.
  	68	No acclimation, flash		Likely	#3 Holder,	Red soda	Straw
  		frozen.		same as	Tank 2	straw, in	performed
  				samples		Aluminum	well. Did
  				above		sleeve, with	not
  				and below		75 ul mylar	explode.
  				this line.		capillary
  						tube, w/
  						clay and
  						critocap
  						with teal
  						cap 1 end,
  						and clay
  						and crito
  						cap other
  						end.
  	69	No acclimation was done		SAP,	#2 holder,	Aluminum	This straw
  		and it was flash frozen. It		BTE, no	Tank 2.	sleeve with	did well
  		was carried to the garage		fructose,		a White	and did not
  		on cold gel packs.		plus		soda straw,	explode,
  				Maple		with 75 ul	but the
  				Tree sap,		capillary	large teal
  				first run,		tube, 1 end	caps on
  				Tree		left open,	the end is
  				number 3.		other end	slow to
  						capped	thaw. The
  						with clay, 1	open end
  						critocap, 1	is key to
  						teal cap.	this
  							success.
  	70	No acclimation was done		SAP,	#2 holder,	Red soda	Straw
  		and it was flash frozen. It		BTE, no	Tank 2.	straw with	performed
  		was carried to the garage		fructose,		holes, plus	well, did
  		on cold gel packs.		plus		plastic	not
  				Maple		poultry	explode.
  				Tree sap,		straw, with
  				first run,		a 75 ul
  				Tree		capillary
  				number 3.		tube, 1 end
  						left open,
  						the other
  						end is
  						clayed, slid
  						into poultry
  						straw with
  						cotton
  						facing
  						down.
  						Straw is
  						ventilated.
  	71	No acclimation was done		SAP,	#2 holder,	Red soda
  		and it was flash frozen. It		BTE, no	Tank 2.	straw with
  		was carried to the garage		fructose,		holes, plus
  		on cold gel packs.		plus		plastic
  				Maple		poultry
  				Tree sap,		straw, with
  				first run,		a 75 ul
  				Tree		capillary
  				number 3.		tube, 1 end
  						left open,
  						the other
  						end is
  						clayed, slid
  						into poultry
  						straw with
  						cotton
  						facing
  						down.
  	72	No acclimation was done		SAP,	#2 holder,	Red soda	Straw
  		and it was flash frozen. It		BTE, no	Tank 2.	straw with	performed
  		was carried to the garage		fructose,		holes, plus	well.
  		on cold gel packs.		plus		plastic
  				Maple		poultry
  				Tree sap,		straw, with
  				first run,		a 75 ul
  				Tree		capillary
  				number 3.		tube, 1 end
  						left open,
  						the other
  						end is
  						clayed, slid
  						into poultry
  						straw with
  						cotton
  						facing
  						down.
  	73	No acclimation was done		SAP,	#1 holder,	Pink soda	Straw
  		and it was flash frozen.		BTE, no	Tank #2	straw, plus	performed
  				fructose,		a plastic	well. It did
  				plus		poultry	not
  				BIRCH		straw, plus	explode.
  				tree sap.		a 75 ul
  				Sucrose		capillary
  				is lower		tube with 1
  				than		end sealed
  				Maple		with clay,
  				tree sap.		with cotton
  						on poultry
  						straw
  						facing
  						down.
  						Number on
  						straw reads
  						67.
  	74	2 samples were		16% MA	Holder #3	Orange
  		produced due to the		used as	in Can #1.	(#68 on
  		volume of the sample.		half of the		straw) and
  		Both were flash frozen.		sample so		Pink soda
  		No gel packs were used.		the final		straws (#
  				concentration		68 on
  				of the		straw), with
  				MA was		a plastic
  				8%.		poultry
  						straw
  						inside, with
  						a 75 ul
  						mylar
  						capillary
  						tube clayed
  						on one
  						end, with
  						cotton on
  						poultry
  						straw
  						facing
  						down.
  	75	Separated into 3 straws		16% MA	#3 Holder	3 green
  		as it was so large. On gel		used as	Tank #1.	soda
  		packs less than 2		half of the		straws, no
  		minutes and then flash		sample so		holes cut in
  		frozen.		the final		them.
  				concentration		Plastic
  				of the		poultry
  				MA was		straws with
  				8%.		75 ul mylar
  						capillary
  						tubes, 1
  						end has
  						clay, the
  						cotton on
  						the poultry
  						straw faces
  						down.
  	76	Acclimated in the fridge	7.5 on pH	Beltsville	#3 Holder	Soda
  		for 10 minutes, DMA	tape after	Turkey	Tank #1.	straw plus
  		added, used bottom of	thaw.	Extender,		plastic
  		fridge at 40 F. AND		no		poultry
  		TURNED DOWN THE		fructose,		straw, plus
  		FRIDGE FOR THE		plus ½%		a 75 ul
  		FIRST TIME so it went		sucrose.		mylar
  		up in temperature. THE				capillary
  		TOP OF THE FRIDGE				tube with
  		WAS AT 35 F. AND THE				clay on 1
  		BOTTOM OF THE				end. With
  		FRIDGE WAS AT 40 F.				cotton on
  		BEFORE I TURNED IT				poultry
  		DOWN. It now sits at 42				straw
  		at the bottom where I				facing
  		am now holding the				down. Not
  		samples so it is 9				ventilated.
  		degrees warmed for				Blue in
  		processing. The				color.
  		samples done from
  		here out are done at a
  		warmer temperature.
  	77	Flash frozen after 10	7.5 pH on	BTE, no	Holder #3	3 soda
  		minutes of acclimation at	pH tape	fructose,	in Can #1.	straws,
  		41 F.	after	plus ½%		light green,
  			thaw.	sucrose,		dark green,
  				and DMA		orange,
  						with a
  						poultry
  						straw
  						inside that
  						and a 75 ul
  						mylar tube
  						inside of
  						that. Green
  						straw was
  						not
  						ventilated
  						and the
  						Orange
  						straw was
  						not
  						ventilated.
  	78	Flash frozen after 10		BTE, no	Holder #3	2 soda	Storage
  		minutes of acclimation at		fructose,	in Can #2.	straws,	with a
  		41 F.		plus Birch		both pink,	Ventilated
  				Tree sap.		both	soda straw,
  						ventilated,	poultry
  						with poultry	straw
  						straw and a	inside that
  						75 ul mylar	[Poultry
  						capillary	straw
  						tube inside	crimped on
  						that. Clay	top end
  						on one end	and cotton
  						leaving it	on the
  						open on	bottom
  						top.	end], with a
  							75 ul mylar
  							coated
  							capillary
  							tube, clay
  							on one end
  							only with
  							the other
  							end left
  							open to
  							LN2;
  							Performed
  							the BEST
  							Keep the
  							material
  							open so
  							they do not
  							explode.
  							All
  							samples
  							stored this
  							way from
  							this point
  							on.
  	79	Sample was not		Birch tree	Holder #3,	2 Soda	Well, no
  		acclimated in the fridge.		sap in	Tank #2.	straws, one	problems.
  		It was mixed, placed in		BTE, No		yellow and
  		tubes, and then put		fructose		one green,
  		between gel packs from				both
  		the fridge, carried out to				ventilated
  		the LN2 can, and flash				with holes;
  		frozen.				with a
  						poultry
  						straw,
  						cotton
  						facing
  						down,
  						crimped on
  						top end;
  						with 75 ul
  						capillary
  						tubes,
  						clayed on
  						one end.
  	80	Acclimated in the fridge		Maple	Holder #3,	2 soda	Well, no
  		for 10 minutes in		tree sap	Tank #2.	straws, one	problems.
  		separate tubes, then		in BTE,		orange and
  		combined and packaged,		no		one pink,
  		then flash frozen		fructose.		both
  						ventilated,
  						with poultry
  						straw
  						inside, with
  						75 ul mylar
  						coated
  						capillary
  						tube inside.
  	81	Acclimated for 10		Maple	Holder #3,	2 Soda	Well, no
  		minutes in the fridge at		tree sap	Tank #2.	straws that	problems.
  		41 F., and then flash		in BTE,		were
  		frozen		no		ventilated,
  				fructose.		with poultry
  						straw
  						inside,
  						cotton end
  						down; 75 ul
  						mylar
  						capillary
  						tube, clay
  						on one end
  	82	Semen and BTE − Fruc, +	pH on pH	Birch tree	2 straws	4 green	Does not
  		½% sucrose	tape after	sap in	in Holder	soda	explode
  		acclimated in separate	thaw was	BTE, No	#6 tank 1,	straws that	but thaws
  		tube from Birch sap BTE,	7.0, 7.2,	fructose	and 2	were	too slowly.
  		at 42 F. in fridge for 10	7.0, 7.5.		straws in	ventilated,
  		minutes, combined, then			Holder #5	75 ul mylar
  		packaged in 4 tubes, 75			tank 1.	capillary
  		ul capillary tube, caulked				tube, inside
  		one end; placed in				poultry
  		poultry straw, cotton				straw,
  		down; inside ventilated				inside
  		soda straw. 4 straws				ventilated
  		made. All green soda				soda straw.
  		straws.
  	83	Semen and BTE − Fruc, +	pH on pH	Maple	2 straws	3 yellow	Good
  		½% sucrose	tape after	tree sap	in Holder	soda
  		acclimated in separate	thaw was	in BTE,	#6 tank 1,	straws that
  		tube from Maple tree sap	7.5-8, 7.0, 7.0	no	and 1	were
  		BTE, at 42 F. in fridge for		fructose.	straws in	ventilated,
  		10 minutes, combined,			Holder #5	75 ul mylar
  		then packaged in 4			tank 1.	capillary
  		tubes, 75 ul capillary				tube, inside
  		tube, caulked one end;				poultry
  		placed in poultry straw,				straw,
  		cotton down; inside				inside
  		ventilated soda straw. 3				ventilated
  		straws made. 3 Yellow				soda straw.
  		straws.
  	84	Semen and BTE − Fruc, +	Mar. 1, 2016	Maple	2 straws	3 pink soda	Tubes
  		½% sucrose	pH on	tree sap	in Holder	straws that	store well
  		acclimated in separate	tape of 7.	in BTE,	#6 tank 1,	were	but it is
  		tube from Maple tree sap	Mar. 6, 2016	no	and 1	ventilated,	best to use
  		BTE, at 44 F. in fridge for	pH on	fructose.	straws in	75 ul mylar	the soda
  		10 minutes, combined,	tape of 7.5.		Holder #5	capillary	straw and
  		then packaged in 3			tank 1.	tube, inside	the mylar
  		tubes, 75 ul capillary				poultry	capillary
  		tube, caulked one end;				straw,	tube only.
  		placed in poultry straw,				inside
  		cotton down; inside				ventilated
  		ventilated soda straw. 3				soda straw.
  		straws made. 3 Pink				One
  		straws.				sample
  						was a
  						capillary
  						tube and
  						soda straw
  						only.
  	85	Semen and BTE − Fruc, +	pH on pH	Maple	2 straws	3 Yellow
  		½% sucrose	tape was	tree sap	in Holder	soda
  		acclimated in separate	7.0, pH on	in BTE,	#6 tank 1,	straws that
  		tube from Maple tree sap	pH tape	no	and 1	were
  		BTE, at 44 F. in fridge for	was 7.0	fructose/	straws in	ventilated,
  		10 minutes, combined,		with 10%	Holder #5	75 ul mylar
  		then packaged in 3		Yolk.	tank 1.	capillary
  		tubes, 75 ul capillary				tube, inside
  		tube, caulked one end;				poultry
  		placed in poultry straw,				straw,
  		cotton down; inside				inside
  		ventilated soda straw. 3				ventilated
  		straws made. 3 Yellow				soda straw.
  		straws. 10% Yolk Added
  		to both mixes.
  	86	All volumes added		Maple	Holder #5,	1 pink soda
  		together, No acclimation.		tree sap	Tank 1.	straw that
  		Flash frozen. 10% Yolk		in BTE,		was
  		Added.		no		ventilated,
  				fructose/		75 ul mylar
  				with 10%		capillary
  				Yolk.		tube, inside
  						a poultry
  						straw,
  						inside the
  						soda straw.
  	87	Acclimated for 10	pH of 7 on	Maple	Holder #6,	Unknown	Stores
  		minutes in the fridge at	pH tape	tree sap	in can #1.	color of	well, but
  		43 F., and then flash	after	in BTE no		soda straw.	thaws too
  		frozen. 10% Yolk	thaw.	fructose/		Was a Pink	slow.
  		added.		with yolk.		ventilated
  						soda straw.
  	88	Acclimated for 10	2016 pH	Maple	Holder #6,	Two pink	Straws
  		minutes in the fridge at	on pH	tree sap	in can #1	soda	work well
  		43 F., and then flash	tape 7, pH	in BTE no	has 2 pink	straws,	but insulate
  		frozen. 10% Yolk	on pH	fructose/	straws,	ventilated,	too well on
  		added.	tape of 7	with yolk.	and	with a 75 ul	thawing.
  					Holder #5,	mylar
  					in can #1	capillary
  					has 1 pink	tube inside
  					straw.	a poultry
  						straw.
  	89	Acclimated for 10	2016 pH	Birch tree	Holder #6	Two	Does not
  		minutes in the fridge at	on tape	sap in	has 1	orange	explode
  		43 F., and then flash	of 7.0	BTE, No	orange	soda	but thaws
  		frozen. 10% Yolk		fructose,	soda	straws,	too slowly.
  		added.		Plus	straw, and	ventilated,
  				YOLK	Holder #5	with 75 ul
  					has 1	mylar
  					orange	capillary
  					soda	tubes
  					straw.	inside
  						poultry
  						straws.
  	90	Acclimated for 10	pH on	Birch tree	Holder #6	Three	Does not
  		minutes in the fridge at	tape of	sap in	has 2	green soda	explode
  		43 F., and then flash	7.0, 7.0,	BTE, no	green	straws,	but thaws
  		frozen. 10% Yolk	7.0.	fructose	soda	ventilated,	too slowly.
  		added.		Plus Yolk.	straws	with 75 ul
  					and	mylar
  					Holder #5	capillary
  					has 1	tubes
  					green	inside
  					soda	poultry
  					straw.	straws.
  	91	Temperature dropped	pH on	MA only	Holder #4,	2 soda
  		only by using gel packs.	tape after		Tank 1. 2	straws, one
  			thaw was		straws,	green and
  			7.5, 7.5		one is	one deep
  					green and	blue.
  					the other	Ventilated
  					deep	with 75 ul
  					purple.	mylar
  						capillary
  						tube inside
  						a poultry
  						straw.
  	92	Temperature dropped		MA only	Holder 3,	1 green	Stores
  		only by using gel packs.			Tank 1, 1	soda straw,	well, but
  					green	ventilated.	thaws too
  					soda	With 75 ul	slow.
  					straw.	mylar
  						capillary
  						tube inside
  						a poultry
  						straw.
  	93	Chilled at 45 F. for 10	pH on	MA only	Holder 4,	3 green
  		minutes and then flash	tape after		Tank 1, 3	soda
  		frozen.	thaw was		green	straws,
  			7.5, 7.5, 7.5.		soda	ventilated,
  					straws all	with 75 ul
  					here.	mylar
  						capillary
  						tube inside
  						a poultry
  						straw.
  	94	Chilled at 45 F. for 10		DMA	Holder #5,	1 orange
  		minutes and then flash			Tank 1.	soda straw
  		frozen.				with 2
  						poultry
  						straws
  						inside of it
  						because I
  						ran out of
  						staples and
  						had 1 soda
  						straw left
  						with a
  						staple in it,
  						marked
  						with # 89
  						on it.
  	95	It was mixed at room	2016 pH 7	Purdy	Holder 5	2 orange	Ventilated
  		temperature with no		10%	and 6,	soda	soda straw
  		acclimation and then		Maple	Tank 1.	straws with	with poultry
  		flash frozen. Lack of		Tree Sap,		75 ul mylar	straw and
  		acclimation reduces cell		326		capillary	mylar
  		survival.		mOsm.		tubes	capillary
  						inside	tube inside
  						poultry	that thaws
  						straws.	too slowly.
  	96	It was mixed at room		Purdy	Holder #	1 light
  		temperature with no		10%	5, Tank 1,	yellow soda
  		acclimation and then		Maple	Light	straw with
  		flash frozen.		Tree Sap,	yellow	75 ul mylar
  				326	soda	capillary
  				mOsm.	straw.	tube inside
  						a poultry
  						straw.
  	97	It was mixed at room	pH of 7 on	Purdy	Holder #	1 pink soda	Stores
  		temperature, sandwiched	pH tape	10%	6, Tank 1.	straw that	well, but
  		between gel packs at 43	after	Maple	Pink soda	was	thaws too
  		F., and then flash frozen.	thaw.	Tree Sap,	straw.	ventilated,	slow.
  				326		75 ul mylar
  				mOsm.		capillary
  						tube, inside
  						a poultry
  						straw,
  						inside the
  						soda straw.
  	98	Purdy formulas begin
  		with # 95.
  	99	It was mixed at room	Feb. 24, 2016	Purdy 20%	Holder #4	3 pink soda
  		temperature, sandwiched	Pink	Maple	has 2 pink	straws that
  		between gel packs at 43	straw, no	Tree Sap,	soda	are
  		F., and then flash frozen.	pH done,	308	straws	ventilated,
  			Second	mOsm.	and	75 ul mylar
  			straw pH		Holder #5	capillary
  			of 7.5,		has 1 pink	tube, inside
  			Last straw		soda	a poultry
  			no pH		straws.	straw,
  			done.			inside the
  						soda straw.
  	100	It was mixed at room	Mar. 6, 2016	Purdy 20%	Three	4 greens	Good,
  		temperature, sandwiched	pH 7 on	Maple	green	soda	stores well
  		between gel packs at 43	tape after	Tree Sap,	soda	straws that	but thawed
  		F., and then flash frozen.	thaw.	308	straws in	are	too slow.
  			Mar. 6, 2016	mOsm.	Holder #6,	ventilated,
  			pH of 7 on		1 straw in	75 ul mylar
  			tape after		holder #5.	capillary
  			thaw.			tube, inside
  						a poultry
  						straw,
  						inside the
  						soda straw.
  	101	It was mixed at room	Mar. 2, 2016	Purdy	Two	2 Yellow	Soda straw
  		temperature, sandwiched	pH of 7 on	10%	yellow	soda	did not
  		between gel packs at 43	pH tape	Maple	straws,	straws that	have
  		F. for 2 minutes, and then	after	tree sap	one in	are	capillary
  		flash frozen.	thaw.		holder #6,	ventilated,	tube in it,
  					and one	75 ul mylar	Lost in the
  					in holder	capillary	tank.
  					#5.	tube, inside
  						a poultry
  						straw,
  						inside a
  						soda straw.
  	102	It was mixed at room	Mar. 2, 2016	Purdy 5%	3 orange	3 orange	Good,
  		temperature, sandwiched	pH of 7;	Maple	soda	soda	stores well
  		between gel packs at 43	Mar. 6, 2016	tree sap.	straws, 2	straws that	but thawed
  		F. for 2 minutes, and then	pH of 7.		in holder	are	too slow.
  		flash frozen. No			#6, and 1	ventilated,
  		acclimation.			in holder	75 ul
  					#5	capillary
  						tube inside
  						a poultry
  						straw.
  	103	It was mixed at room	7.5 and 7,	Purdy	4 green	4 green	Good
  		temperature, with no	7, 7.	10%	soda	soda
  		acclimation, and then		Maple	straws, 2	straws that
  		flash frozen.		tree sap,	straws in	were
  				plus 5%	holder #5	ventilated,
  				(13 ul) of	and 2	75 ul mylar
  				DMA.	straws in	capillary
  					holder #6.	tube, inside
  						poultry
  						straw,
  						inside
  						ventilated
  						soda straw.
  	104	It was mixed at room	7	Purdy	Yellow	Yellow	Good
  		temperature, with no		10%	straw in	soda straw
  		acclimation, and then		Maple	holder #5	that was
  		flash frozen.		tree sap		ventilated,
  				plus		75 ul mylar
  				Arabogalactin		capillary
  				and		tube, inside
  				then 12%		poultry
  				MA.		straw,
  						inside
  						ventilated
  						soda straw.
  	105	Chilled 15 minutes and	7.51 and 7.23.	2015	2 straws	Mylar	Stores well
  		then second diluent	(adjusted	extenders	in Holder	capillary	and thaws
  		added. Packaged quickly	wrong	that have	#6, 1	tube,	well.
  		and then flash frozen.	and bad	sap in	straw in	caulked on
  			for	them.	Holder #5	one end,
  			motility).	Second		inside a
  				diluent		small
  				had pH		ventilated
  				adjusted		soda straw.
  				up with
  				bicarb.
  	106	Acclimated in the fridge	7.51 and 7.23.	2015	2 straws	Mylar	Stores well
  		at 42 F. for 15 minutes	(adjusted	extenders	in Holder	capillary	and thaws
  		and then flash frozen.	wrong	that have	#6 and 1	tube,	well.
  			and bad	sap in	straw in	caulked on
  			for	them.	Holder #5.	one end,
  			motility).	Second		inside a
  				diluent		small
  				had pH		ventilated
  				adjusted		soda straw.
  				up with
  				bicarb.
  	107	Acclimated 16 minutes at	pH 7.51	2015	2 straws	Mylar	Stores well
  		42 F. and then packages	then	extenders	in Holder	capillary	and thaws
  		into 3 straws and flash	pH 7.23	that have	#6 and 1	tube,	well.
  		frozen.	(adjusted	sap in	straw in	caulked on
  			wrong	them.	Holder #5.	one end,
  			and bad	Second		inside a
  			for	diluent		small
  			motility).	had pH		ventilated
  				adjusted		soda straw.
  				up with
  				bicarb.
  	108	Acclimated 16 minutes at	pH 7.51	2015	2 straws	Mylar	Stores well
  		42 F. and then packages	then	extenders	in Holder	capillary	and thaws
  		into 3 straws and flash	pH 7.23	that have	#6 and 1	tube	well.
  		frozen.	(adjusted	sap in	straw in	caulked
  			wrong	them.	Holder #5.	one end
  			and bad	Second		with
  			for	diluent		ventilated
  			motility).	had pH		soda straw.
  				adjusted
  				up with
  				bicarb.
  	109	Acclimated 16 minutes at	pH of 7.51	2015	2 straws	Mylar	Stores well
  		42 F. and then packages	then pH	extenders	in Holder	capillary	and thaws
  		into 4 straws and flash	of 7.23	that have	#6 and 2	tube	well.
  		frozen.	(adjusted	sap in	straws in	caulked
  			wrong	them.	Holder #5.	one end
  			and bad	Second		with
  			for	diluent		ventilated
  			motility).	had pH		soda straw.
  				adjusted
  				up with
  				bicarb.
  	110	Acclimated 16 minutes in	pH of 7.51	2015	2 straws	Mylar	Stores well
  		the fridge at 42 F. and	then pH	extenders	in Holder	capillary	and thaws
  		then flash frozen.	of 7.23	that have	#6 and 1	tube	well.
  			(adjusted	sap in	straw in	caulked
  			wrong	them.	Holder #5.	one end
  			and bad	Second		with
  			for	diluent		ventilated
  			motility).	had pH		soda straw.
  				adjusted
  				up with
  				bicarb.
  	111	Acclimated for 18	pH of 7.51	2015	2 straws	Mylar	Stores well
  		minutes at 42 F. in the	then pH	extenders	are in	capillary	and thaws
  		fridge and then flash	of 7.23	that have	Holder #6	tube	well.
  		frozen.	(adjusted	sap in	and 1	caulked
  			wrong	them.	straw is in	one end
  			and bad	Second	Holder #5.	with
  			for	diluent		ventilated
  			motility).	had pH		soda straw.
  				adjusted
  				up with
  				bicarb.
  	112	Acclimated on gel packs	7.4 then	BTE	1 straw in	Mylar	Caulk
  		at 42 F. for 16 minutes.	7.23 both	minus	holder #5	capillary	tends to
  		Then flash frozen. One	2015 diluents	fructose,	and 2	tube	come out
  		sample got caught on the	with sap	plus ½%	straws in	caulked	on thaw
  		holder and stayed above	that both	sucrose	holder #4.	one end	due to LN2
  		the LN2 and was not	had pH	with pH		with	pressure
  		flash frozen. The other	adjustments	adjusted		ventilated	inside the
  		samples in the group	that	with		soda straw.	capillary
  		were flash frozen and	went bad.	bicarb			tube.
  		died.		from 7.51
  				to 7.4.;
  				Then
  				added
  				BTE −
  				Fructose +
  				Maple
  				Tree Sap
  				tree #3
  				with pH
  				adjusted
  				with
  				bicarb to
  				7.23.
  	113	Acclimated for 16	7.4 then	BTE	1 straw in	Mylar	Stores well
  		minutes in the fridge at	7.23 both	minus	holder #5	capillary	and thaws
  		42 F. and then flash	2015 diluents	fructose,	and 2	tube	well.
  		frozen.	with sap	plus ½%	straws in	caulked
  			that both		holder #4.	one end
  			had pH	sucrose		with
  			adjustments	with pH		ventilated
  			that	adjusted		soda straw.
  			went bad.	with
  				bicarb
  				from 7.51
  				to 7.4.;
  				Then
  				added
  				BTE −
  				Fructose +
  				Maple
  				Tree Sap
  				tree #3
  				with pH
  				adjusted
  				with
  				bicarb to
  				7.23.
  	114	Acclimated for 16	7.4 then	BTE	1 tube in	Mylar
  		minutes in the fridge at	7.23 both	minus	Holder #5	capillary
  		42 F. and then flash	2015 diluents	fructose,	and 1	tube
  		frozen.	with sap	plus ½%	tube in	caulked
  			that both	sucrose	Holder #4.	one end
  			had pH	with pH		with
  			adjustments	adjusted		ventilated
  			that	with		soda straw.
  			went bad.	bicarb
  				from 7.51
  				to 7.4.;
  				Then
  				added
  				BTE −
  				Fructose +
  				Maple
  				Tree Sap
  				tree #3
  				with pH
  				adjusted
  				with
  				bicarb to
  				7.23.
  	115	Acclimated 20 minutes
  		and then flash frozen.
  	116	Acclimated 15 minutes	pH 6.27	Amur	3 straws	Mylar	Stores well
  		and then flash frozen.	then	Maple +	in Holder	capillary	and thaws
  			pH 6.74.	BTE −	#3.	tube	well.
  				Fruc with		caulked
  				adjusted		one end
  				pH of 6.74		with
  				with		ventilated
  				Glutathione +		soda straw.
  				NN-
  				Bis . . . Sulfonic
  				Acid.
  	117	Acclimated 15 minutes	pH 6.27	Amur	4 straws	Mylar	Stores well
  		and then flash frozen.	then	Maple +	in Holder	capillary	and thaws
  			pH 6.74.	BTE −	#3.	tube	well.
  				Fruc with		caulked
  				adjusted		one end
  				pH of 6.74		with
  				with		ventilated
  				Glutathione +		soda straw.
  				NN-
  				Bis . . . Sulfonic
  				Acid.
  	118	Acclimated semen in its	pH 6.48	BTE −	2 straws	Mylar
  		own tube for 5 minutes		Fructose +	in holder	capillary
  		and then added in the		Maple	#3.	tube +
  		Maple tree sap tree #3		Tree #3		Critocap +
  		(2015). Then slowly		sap,		Blue cap +
  		lowered into LN2.		2015.		Ventilated
  				(original		soda straw.
  				6.48 pH
  				from
  				2015)
  	119	Put in holder #5 above	7.51. then 6.48	BTE −	#5	Mylar	Caulk
  		liquid nitrogen vapors for		Fructose +		capillary	tends to
  		10 seconds and then		½%		tube,	come out
  		flash froze with slow		Sucrose		caulked on	on thaw
  		immersion.		(pH 7.51),		one end,	due to LN2
  				then BTE −		inside a	pressure
  				Fructose +		small	inside the
  				Maple		ventilated	capillary
  				Tree #3		soda straw.	tube.
  				sap,
  				2015. (pH
  				6.48).
  	120	Acclimated 15 minutes at	7.51 then 6.48	BTE −	4 straws	Mylar	Caulk
  		42 F. then hung over the		Fructose +	in Holder	capillary	tends to
  		vapors 15 seconds and		½%	#3.	tube,	come out
  		then slowly lowered into		Sucrose		caulked on	on thaw
  		LN2.		(pH 7.51);		one end,	due to LN2
  				then BTE −		inside a	pressure
  				Fructose +		small	inside the
  				Maple		ventilated	capillary
  				Tree #3		soda straw.	tube.
  				sap,
  				2015.
  				(pH 6.48).
  	121	Acclimated 15 minutes at	7.51 then 6.48	BTE −	2 straw in	Natellson
  		42 F. then hung over the		Fructose +	Holder #3.	Capillary
  		vapors 15 seconds and		½%		tube with 1
  		then slowly lowered into		Sucrose		cap in a
  		LN2.		(pH 7.51);		large
  				then BTE −		ventilated
  				Fructose		soda straw.
  				plus		(Orange).
  				Maple
  				Tree sap
  				#3 2015
  				(pH. 6.48)
  	122	Acclimated 15 minutes at	pH 7.51	BTE −	1 straw in	Natellson
  		42 F. then hung over the	then	Fruc +	Holder #3.	capillary
  		vapors 15 seconds and	pH 6.48.	½% Suc		tube + cap +
  		then slowly lowered into		(pH 7.51);		Large
  		LN2.		then BTE −		ventilated
  				Fruc +		soda straw
  				1st Run		(Green).
  				Maple
  				tree sap.
  				2015
  	123
  	124
  	125
  	126	Acclimated for 15	pH 7.51	BTE −	Three	Mylar	Caulk
  		minutes at 42 F. in the	then	Fructose +	straws in	capillary	tends to
  		fridge, and then flash	pH 6.48.	½%	Holder #5,	tube	come out
  		frozen in LN2.		Sucrose	1 Straw in	caulked	on thaw
  				2015	Holder #4.	one end	due to LN2
  				(pH 7.51);		with	pressure
  				then		ventilated	inside the
  				added		soda straw.	capillary
  				BTE −			tube.
  				Fructose +
  				Maple
  				Tree Sap
  				tree #3
  				2015
  				(pH 6.48)
  	127		pH 7.56		#4	Mylar
  			then			capillary
  			pH 6.48			tube
  						caulked
  						one end
  						with
  						ventilated
  						soda straw.
  	128	Acclimated for 15	pH 7.56	BTE −	3 straws	Mylar	Caulk
  		minutes then suspended	then	Fructose	in Holder	capillary	tends to
  		over LN2 vapors for 15	pH 6.48	plus 1st	#4.	tube	come out
  		seconds and then slowly		run		caulked	on thaw
  		lowered into LN2.		Alaska		one end	due to LN2
  				Birch		with	pressure
  				(pH 7.56)		ventilated	inside the
  				and then		soda straw.	capillary
  				BTE −			tube.
  				Fructose +
  				Maple
  				Tree #3
  				2015
  				(pH 6.48)

Claims (20)

I claim:

1. A method of cryogenically preserving sperm comprising:

a. combining sperm to be cryogenically preserved and a composition that comprises (1) a cryoprotectant, comprising one or more tree saps; and (2) an extender medium to produce a sperm/medium combination; and

b. subjecting the combination to conditions that result in cryopreservation of sperm, thereby producing a cryopreserved combination that comprises cryopreserved sperm,

wherein the one or more tree saps is derived from cold-hardy trees.

2. The method of claim 1 wherein the cryopreserved sperm of step (b) demonstrates survival greater than 50% after thawing.

3. The method of claim 1 wherein the cryopreserved sperm of step (b) demonstrates motility greater than 30% after thawing.

4. The method of claim 1 wherein the one of more tree saps is the only cryoprotectant.

5. The method of claim 1 wherein an additional cryoprotectant is added.

6. The method of claim 1 wherein the sperm is avian sperm.

7. The method of claim 6 wherein the sperm is derived from the Northern goshawk (Accipiter gentilis).

8. The method of claim 1 wherein the sperm is derived from a non-human mammal.

9. The method of claim 8 wherein the sperm is derived from an animal type selected from the group consisting of canine, avian, cattle, porcine and equine.

10. The method of claim 1 wherein the one or more tree saps is a first run sap.

11. The method of claim 1 wherein the extender medium does not contain fructose.

12. The method of claim 1 wherein the method comprises the additional step of subjecting the combination to a temperature between −80° C. and −198° C. for a period of at least one day.

13. The cryopreserved combination resulting from the method of claim 1.

14. The cryopreserved combination resulting from the method of claim 2.

15. The cryopreserved combination resulting from the method of claim 3.

16. A method of fertilizing an egg cell comprising the step of thawing a cryopreserved combination produced by the method of claim 1, and introducing the combination to an unfertilized egg cell, wherein the egg cell becomes fertilized.

17. The method of claim 16, wherein the egg cell is an avian egg.

18. The method of claim 16, wherein the egg cell is a mammalian egg.

19. A composition comprising tree sap, semen, and extender medium, wherein the tree sap is a cold-hardy tree sap.

20. The composition of claim 19, wherein the sap is at least 50% by volume of the composition.