Improving Yak Artificial Insemination Conception Rate and Providing Data for Informed Decision-making for Improving Farm Sustainability

A. Semen Collection

One of the key things we have learned in this project based on the fifteen yak bulls we worked with, is that yak sperm is very ambient temperature sensitive.  Like beef bulls, and as shown in the FS21-335 final report, yak semen quality in terms of motility and morphology is dependent on the ambient temperature during the sixty days required for it to develop. In Eastern Kentucky, from the end of April to at least mid-October, even though yak bulls can technically get yak cows pregnant in the field, the quality of their sperm is very low in terms of percent motility and percent with good morphology…Too low to consider collecting for extending and preserving for artificial insemination. Over the past four years we have to use a threshold of 80% motility and 80% morphology at collection in order to have a sufficient post thaw viable sperm assuming the initial concentration was good.  Furthermore, if a bull was sick or had other sources of stress during sixty days of sperm development, no matter what the ambient temperature was during that period, the quality of the semen can be negatively effected. 

Note: The semen collected from four yak bulls at the end of October of 2025 had very low motility and morphology in three of the four bulls due to the extended high ambient temperature through the summer into the fall. When we collected again, in November, all the bulls’ sperm had a motility and morphology greater than 80%. 

Over the past thirty plus years the development of quality beef bulls has included scrotal size, which is a heritable trait, as a key EPD.  This is because the larger the scrotal size the greater the quantity and quality of the ejaculate as well as impacting the reproductive maturation of the heifers sired.  This kind of research has not been done with yaks in the United States. In fact there has been no emphasis in breeding programs to increase scrotal size – and therefore, the quantity and quality of yak semen.  However, based on the more than fifteen yak bulls we selected to collect semen from over the past two projects: If a yak bull’s testicular circumference was less than 27 centimeters, its semen was never good enough to collect for extending in terms of sperm concentration and semen volume collected.

When collecting yak semen, and all that takes place between collecting it and getting it in a 99-100 degree water bath to await being extended, TEMPERATURE CONTROL IS EVERYTHING.

Given that yak bull semen had to be collected between late October (at the earliest) and the end of April due to the impact of ambient temperature on the quality of a bull’s, we were always collecting semen in cool to cold weather. And given that yak bull semen is 101 degrees in temperature, even in the summer a semen collection tube would have a far lower temperature than the bull’s semen: Let alone the ambient temperature on a late fall or winter day. Cold kills semen.  So:

• The greater the temperature difference between the semen collection tube and the temperature surrounding the semen collection tube until the semen is placed in a 99-100 degree water bath; and,
• The amount of time the semen is in a collection tube with a temperature of less than 99-100 degrees
  • => The more yak sperm will die before the extending process is even started.

In our situation, the semen was in the collection tube for minutes in the outside ambient air during the motility measurement and then being transported to a heated room (only in the 70s's) where it was then placed in a 99F water bath.

Holding the collection tube in one’s hand or gloved hand during collection and then holding it inside one’s coat (The traditional method) didn’t keep it warm enough to prevent significant amounts of sperm from dying.

Furthermore, at the other end of just getting the collected semen to the water bath, the now surviving sperm is cooler than the water bath. So when it is suddenly put in the water bath more sperm die from the heat differential.

The solution was quite simple. A simple chemical toe warmer was wrapped around the collection tube prior to collection to bring it up close to 100 F and then was protected in all the traditional methods as well to keep it as warm and protected as possible. Just this change made a significant difference in the final amount of viable extended sperm produced.

Remember: You will have loss at every step of the process.

• Collecting from the bull
• Every second the semen is at less than 99-100F until it is in the water bath.
• During the extending process every time it is out of the water bath until it has been extended since the room it is being extended in is at a temperature far less than 99-100F
• After the extended semen has cooled and is ready for loading straws: Here it is the reverse problem. You do not want the extended semen to warm up during the straw loading process.
• And after the straws have been frozen in the liquid nitrogen. You don’t want them to thaw while they are loaded in goblets and canes and finally placed in a tank of Liquid Nitrogen.

Each step in the process will have loss. The key is to minimize the loss at every step

The steps in semen collection we used and recommend are as follows:

1. The bull is weighed too see if there could be a health issue that could impact perm quality and for record keeping
2. The bull then advances to the squeeze chute where it is constrained.
3. The collection equipment is ready and a toe warmer designed to heat to 102F is wrapped around the collection tube which is then put in an inside pocket of the person who will hold it during collection.
4. The yak's temperature is taken - again if out of the normal range could indicate a problem with the sperm,
5. The yak's scrotal circumference is measured and recorded. If the scrotal circumference is less than 27cm, the quality of the sperm: concentration, motility and morphology may not be high enough to continue.
6. During this time the toe warmer applied to the collection tube should have brought its’ temperature close to 100F.
7. The bull's Cowper's and  Prostrate glands and the Seminal vesicle are very gently manually massaged for approximately two minutes.

The individual with the collection tube needs to be prepared to collect semen at this point since some yak bulls will ejaculate during the massage.

8. A small bovine electro-ejaculator is lubricated and inserted and run on automatic intensity advancement. [This is like an internal muscle stimulator TENS unit for pain management.]
9. Once the bull has settled down and developed a rocking motion the toe warmer encased collection tube should be held in the collector’s hand protecting it and in a position to receive the tip of the bull’s extended penis.
10. Once the bull has deposited sufficient ejaculate in the collection tube the electro ejaculator is turn off and the collection tube is taken as protected as possible to have two pipettes ready to remove a drop of semen for a motility check by microscope and for a morphology evaluation. KEEP THIS TIME TO A MINIMUM. Once the drops have been removed the collection tube is taken to the water bath where it will be maintained at 99-100F.

• NOTE: When doing the motility check on the microscope, be sure the slide and cover have been warmed on a slide warmer before placing the drop of semen from the collection tube on the slide. The time between extracting a drop of the semen with the pipette until viewing by the microscope needs to be as fast as possible to reduce sperm death due to cold. The drop of semen taken for morphology evaluation doesn’t matter as it is the physical shapes of the sperm (eg, heads and tails) that matters.

• NOTE: The collection tube had the toe warmer around it and the collector's hand around it to keep it warm from before the electro-ejaculator cycle was started to when the semen was put in the water bath. The time from the collection to the water bath must be kept at a minimum.

KEY SEMEN COLLECTION GUIDELINES:  

1. KEEP SEMEN AS CLOSE TO 100F AS POSSIBLE DURING COLLECTION AND UNTIL IT IS PUT IN THE WATER BATH.
2. IF MOTILITY OR MORPHOLOGY ARE LESS THAN 70%, THE SEMEN IS NOT GOING TO EXTEND WELL. Note, if the motility or morphology are low it does not mean the bull is "bad". It could be the result of the bull not having ejaculated for quite a while. We have learned to let the bulls we intend to use for AI in with several cows in estrus so they can have done some breeding prior to collection. A low motility and/or morphology can also indicate the bull was under some kind of stress during the prior sixty days while the semen collected was being formed or that that there is a health/nutritional issue.

B. Extending Yak

 SemenIn this project we used a number of different extenders: TRIS extenders prepared by Dr, Lehmkuhler using formulas that had been successfully used for years with beef cattle and two purchased extenders from IMV Technologies: Optidyl – an egg based extender with powdered egg yolk included and BullXcell - an egg based extender which required the addition of egg yolk. In our use of the different extenders the egg based extenders (ones where egg yolk was added or that contained a powdered yolk in them) worked the best.

Note: With respect to adding egg yolk, the Researchers at the ICAR - National Research Centre on Yak in India were very clear about using fresh eggs. While eggs purchased at the grocery are fresh enough for consumption, they may not be fresh enough for making a high quality extender. Using the simple "how an egg sinks" method on eggs purchased at a grocery revealed than many were far from “fresh”. During this project we switched to farm eggs layed within twenty-four hours of making the extender verses store bought organic eggs used in the previous project FS21-335. The extender we have made under this project using truly fresh eggs  has been much improved in terms of post thaw motility which, in part, could be partially due to egg "freshness".  [Note: It is in the little details that the quality of the extended semen can be greatly impacted.]

Note: Making the BullXcell – Adding Fresh Egg Yolk

• In using a fresh egg yolk we first make sure the shell is clean using distilled water and then break the egg shell keeping the yolk inside it and drain off as much of egg white as possible. Once this is done we roll the yolk out of the shell on paper towels and roll it around on the towels so they can capture the fluid on the exterior of the yolk leaving a clean "dry" yolk. We then used a pipette to pierce the yolk and withdraw the amount needed from within.
• A warmer with a magnetic stirrer was used to mix the extender and added egg yolk. It is important that the prepared extender be a homogenous solution.

Note: It is important to note that some extenders will go bad within a certain amount of time.  For example the Bullxcell Extender requires that the semen be added within ten minutes of making.

Key Steps in Extending the Collected Semen

• The collected semen needs to be placed in the water bath upon receiving.
• Once the collected semen has been put in the water bath a sample is taken to determine the sperm concentration per ml using an Accu-cell Bovine Photometer. Note: This device gives a sperm count per ml but does not differentiate between live and dead sperm. Therefore, if the concentration is 400 Million sperm/ml and the motility is 75%, one would assume a live sperm count of 300 Million sperm/ml.
• Once the concentration of sperm is known the amount of extender needed to protect the semen can be calculated. During the past project FS21-335 we extended to achieve a sperm concentration of 100 Million total (alive and dead) sperm per ml in order to try to account for motility and morphology so that the living sperm concentration in the extended semen would be at least 60 Million sperm/ml. To achieve this concentration required we often had to use the minimal amount of extender allowed: a 1:1 semen to extender mix.

Using this small amount of extender may have not been enough to properly protect the yak semen during freezing and later thawing to check post thaw motility and contributed to the low post thaw motility numbers we had been achieving during the FS21-335 project

In this project we have experimented with using extended total (alive and dead) sperm concentrations of 30 Million, 45 million and 60 million per ml. Over the six times we collected and extended semen the 30 Million and 45 Million total sperm per ml post thaw numbers have been excellent and better than the 60 Million total sperm /ml concentration post thaw results: Indicating that the reduced amount of extender needed for a concentration of 60 million sperm did not provide adequate freezing and later thawing protection for the sperm.

• The extender being used should be prepared and placed in the water bath to bring it up to the same temperature as the semen to be extended.
• Recall some extenders have a time limit within which the semen be added so be sure everything is ready once the time limited extender has been prepared and warmed.
• Remember that when you add the proper amount of extender for the desired concentration to the semen that you will be removing both the semen and extender from the water bath to extend. This should be done as quickly as possible to avoid cooling the unprotected sperm . Once combined the extended semen should be placed back in the water bath until it is time to cool the extended semen for packaging (in straws and frozen).

The following are the post thaw results of the three processed semen collections during the first year of Project  FS23-352 using the two different IMV Technologies egg yolk based extenders: BullXcell (fresh egg  yolk added) and Optidyl (egg yolk already contained in the premixed powdered extender). 

Bull               Extended  sperm              Freeze #           BullXcell post thaw      Optidyl post thaw   

                                Concentration                                                  motility                          motility

Bull #47     30 million/ml                               1                              45-50%                              35%

Sad Eyes     45 million/ml                              1                                  55 %                               40%

Phantom     30 million/ml                             1                                  35 -40%                         30% 

Phantom      45 million/ml                             1                                   45%

Phantom     60 million/ml                           1                                   30%  66 minutes post thaw

Bull #47     45 million/ml                               2                              30%                                    25%

Sad Eyes     45 million/ml                              2                                50 %                                45%         

Phantom     45 million/ml                             2                                45%                                  35%

Bull #47     30 million/ml                               3                             40%                                                  

Bull #47     45 million/ml                               3                                                                           35%

Sad Eyes     30 million/ml                              3                                                                            45%

Sad eyes      45 million/ml                              3                             45%

Phantom      30 million/ml                             3                              40%                                      45%  

• Note: All post -motilities were estimated by two researchers viewing sperm motility using a microscope

=>In this case the post thawed extended semen of two straws for each bull were mixed together and evaluated using the Computer Assisted Semen Analyzer.

=> Since whether a concentration of 30 or 45 million sperm per ml was determined to not be a factor in the post thaw motility, concentrations of 45M sperm/ml were used.

 =>Column 3 [ thawtime] is the time motility was measured relative to being thawed:

0 -thaw time is 0 hours post thaw  - when the frozen semen was first thawed

3 – thaw time is three hours post thaw

=>Total motility is the percentage of sperm actively moving in any direction

=>Progressive motility is the percentage of sperm moving forward in a linear or slightly curved path

=>Normal is the percentage of all of the sperm (dead and alive) without defects. Based on a sample of 100.

=>Primary is the percentage of all of the sperm with abnormally shaped heads, midpieces (corkscrew), or    tails.  These defects occur during spermatogenesis. Based on a sample of 100.

=> Secondary is the percentage of all of the sperm with defects that occur after formation typically during  transport and storage in the epididymis or during ejaculation.  Based on a sample of 100.

As an example consider Phantom’s Bullxcell extended semen results. All of the samples in the chart had a concentration of 45 million sperm/ml when extended. 

• 1% of the sperm in this thawed semen sample were normal which is just over 40 million/ml. Remember this 40 million includes live and dead sperm. The dead sperm in the initial collection were never “sifted out” to they are part of the semen contained in the packaged frozen extended semen.
• The Total Motility (number of living sperm) upon thawing was 40.29% of the 45 million which is 18.1 million sperm.  So, at insemination roughly 18.1 million sperm would be alive.
• The Progressive Motility, the percent of sperm that were moving in a forward direction (linear or slight arc) was roughly 15.43 % or 6.94 million sperm at the moment of insemination.
• Three hours later, the number of living sperm was 33.64% of the original 45 million sperm which is just over 15 million sperm still alive. During actual insemination there would be some further loss due to internal environment of the cow’s cervix/uterus where the sperm were deposited at insemination.
• The number of sperm that would still moving in a forward (linear or slight arc) direction apart from environmental losses after three hours would be 20.26% of the 45 million which is just over 9 million sperm

NOTE:  The “increase  in progressively motile sperm” at three hours post thaw can be partially due to the sperm having had more time in a warm environment since post thaw to adjust since the immediate post thaw measurement. The major difference is due to how the test is run: The post thawed sperm is placed in a water bath and a sample is taken immediately and evaluated for progressive motility. Three hours a later a second sample is taken from the original post thawed semen in the water bath and evaluated for progressive motility: Not the original sample evaluated three hours later. The fact that two different samples (same extended semen, but within the thawed semen there is not uniformity of quantity and quality of sperm in terms of motility and progressive motility) were taken gave rise to an unexpected increase in the three hour progressive motility as compared to the one hour progressive motility

NOTE: Look at what happens when Optidyl is used to extend Pantom’s semen.  After three hours the progressive motility is 1.2 % - a half million sperm not counting losses due to the internal environment.

C. Cooling the Extended Semen

Once the semen has been extended the next step is to cool it down to 5C (41F) at the recommended cooling rate of  0.25C per minute. 

To cool the semen to 5C we use a two cubic foot laboratory refrigerator which has a digitally controlled temperature. There was no way to set a cooling rate so what we had to do was determine its cooling rate by placing different volumes of 37C (water bath temperature) water in the refrigerator precooled to 5C and using a digital thermometer with a wire probe record how the interior temperature dropped over time: In reality we were determining the volume of the bath water we needed to have in a beaker with the extended semen so that the total extended semen and bath water would cool at 0.25C/min. Once this “total volume” which would cool at .25 C/minute was determined, the volume of bath water added to the total extended semen was the “volume of water which would cool at ).25C/min minus the volume of extended semen. The total cooling time was (37C-5C)/0.25/min = 128 minutes.

• NOTE: IN determining the volume which would cool at 0.25C/min. we did not have to wait the entire time for the refrigerator to cool to 5C but only long enough to see how many degrees it was cooling per minute once a constant cooling rate developed.

Once we had a volume of water at 37C that cooled at approximately 0.25C per minute we were done. In our case it was 400 ml. Knowing this, for example if we were going to cool a total of 40 ml of extended semen we would put its container in a beaker of 360ml of water for cooling in the refrigerator.

NOTE: We chose not to package the extended semen before cooling because to do so would require us to remove the semen from the bath water temperature of 99F (37C) into the air temperature of the room we worked in which would result in an uncontrolled cooling rate for the first twenty five or more degrees (F): From 99F to room temperature F.

We set the refrigerator in a cold room we constructed which could be cooled to 5C (41F). This way the chilled semen could be removed from the refrigerator, and packaged in straws, and then frozen in the cold room without “warming the cooled semen due to a higher room temperature.

The following took place in our cold room:

• Cooling the semen in the laboratory refrigerator at a rate of 0.25C/min
• Filling 0.5cc straws with the chilled semen. [Note the straws needed to be in the cold room while the semen was being cooled so they would be at 5C as well.] The straws were filled using a “semen straw filling nozzle” which allows fifteen straws to be filled simultaneously.

=>NOTE: Different colored straws were used for each bull. This is important as well as coding by goblet color and labeling the top of the canes since when selecting specifics straws from the Liquid Nitrogen storage tank to use for insemination it is critical that the straws brought out of the Liquid Nitrogen that are not going to be used are out of the LN for as short a time as possible to minimize thawing and then refreezing the thawed sperm which would kill them.

• After filling the straws an air pocket at the open end of each straw was made using a using a “semen straw comb”
• The straws were then sealed using glass sealing balls that had been warmed on a coffee cup warmer. The warming of the sealing balls made the “pushing the straw down over the sealing ball” easier.

=>NOTE: Glass sealing balls were used rather than metal sealing balls since they seemed to hold their position better than the metal balls when placed in Liquid Nitrogen.

• After all the extended semen had been sealed in straws they were placed on freezing racks and cooled in the refrigerator at 5C for an additional three hours.

 After the additional 3 hour cooling the straws are frozen in Liquid Nitrogen, placed in goblets and canes, and then placed in a LN semen storage tank..

NOTE:  Loading straws after extending before cooling verses loading after cooling in the cold room.

Semen straws can be filled after extending rather than in the cold room: The extender will protect the sperm from the change in temperature from the warming bath 99F to the ambient room temperature.

• Once you remove the extended semen from the bath what you don’t want to do is have rapid decreases in temperature or raise the temperature of the extended semen. If there is a temperature change it must be to a lower temperature at a rate less than 0.25C/min. Increasing the temperature of the extended semen will damage the sperm.

The extended semen is then cooled in the straws to 5C at a rate averaging about 0.25C/minute and then keep it at 5C for an additional three hours before freezing.

First: Compute 400ml – the total volume of extended semen in the straws (# of straws x .9 x straw volume). The .9 is added to account for the reduction in the amount of semen in each straw due to the air bubble created at the open end.  THIS IS THE AMOUNT OF WATER THAT MUST BE COOLDED ALONG WITH THE EXTENDED SEMEN.

Second: Since you want the extended semen in the straws to cool at the same rate as the water you are colling with the straws, depending on the number of straws you are cooling, sandwich the straws in a sealed baggie between two baggies partially filled with water, the temperature of the room the straws were filled in, totaling the calculated amount of water (one below the straws and the other on top).  The assumption is that by the time the straws are filled the extended semen has cooled to the temperature of the room in which the straws were filled.  The straws should be placed in a sealed baggy between the two baggies containing water to prevent them from getting wet in case one of the water-filled baggies leaks. 

NOTE: One must be sure that the water-filled baggies above and below the baggie with the semen straws surround all of the straws with water: If not, the extended semen in the straws that are exposed to the air will cool too quickly and be damaged.

We experimented with two methods of filling the straws:

• Filling the straws with extended semen room the water bath (37C=99F) in the room and then placing them in a baggie and sandwiching them between two baggies with water the temperature of the room the straws were filled so that the total volume of water in baggies plus (the number of straws x 0.9 x volume of a straw) = 400ml and then cooling them in the refrigerator at 0.25C/min to 5C  followed by three  hours at 5C in the refrigerator; and,  
• First cooling the warm extended semen at 0.25C to 5C (128 minutes), packaging the chilled semen in the straws and then cooling the straws at 5C for an additional three hours.

We tested the post thaw motility results of extended semen packed both ways and found that cooling the extended semen and then packaging in straws verses packaging the extended warm semen at room temperature followed by cooling. By microscopic examination of the thawed semen,  the researched believed the extended semen which was packaged in straws  at 5C had slightly higher motility.

D. Freezing the cooled straws

Once the straws cooled for the additional three hours at 5C the extended semen is ready to be frozen in Liquid Nitrogen.

In this project we used two freezing techniques:

FIRST SYSTEM: This was the same technique used in Project FS21-335. We had a simple pulley system to control the descent of the freezing rack holding the semen straws into a Styrofoam container (21x13x10.5 inches (inside LxWxH) with 1.5 inch thick walls and a removable friction fit cover) into which we had poured enough liquid nitrogen to completely submerge the freezing rack and straws. The system was a hand crank operate pulley which lowered what looked like the lift system on the front of a forklift made of wood with two right angle brackets large enough for the freezing rack to securely sit on as shown.

A digital thermometer that could read below -140 C and had a wire temperature probe which we could attach to the rack without the exposed probe wire touching the rack was used to monitor the temperature of the straws as it was lowered according to the following protocol:

Starting temperature  5C => the brackets holding the freezing rack need to be able to raise approximately five inches above the walls of the Styrofoam box to keep the rack above the LN vapor in the box in order to start the process at 5C.

1. Five minutes to go "smoothly" from 5C to -10C
2. Four minutes from -10C to -40C
3. Two minutes from -40C to -100C
4. Three minutes from -100C to -140C
5. We held the rack at -140C for one minute and then plunged it into the liquid nitrogen.

The closer the rack was to the liquid nitrogen the more a slight lowering would greatly decrease the temperature. The Temp vs. Time graph shown is from the first three of five times we froze the semen using this protocol.

NOTE that even though the descents were not smooth and had significant differences in rate of decent at each step, all of the resulting post thaw motilities were very close to one another.

SECOND SYSTEM: To test the necessity of a controlled temperature descent we used a second system to freeze the semen, This system consisted of a rectangular insulated metal container 10x10x8 inches which was filled two-thirds full of liquid nitrogen. A small rack that could hold about twenty semen straws was mounted on a rectangular metal frame which was mounted on two Styrofoam pontoons that would float the rack carrying the straws on the liquid nitrogen. The frame was designed to set how high  the straws would be above the liquid nitrogen. The adjustment was from 2 to 5 centimeters. The temperature probe of the digital thermometer was attached to the rack holding the straws in order to monitor the temperature of the air surrounding the straws.

Once the frame was adjusted to the desired height the straws should be above the liquid nitrogen, the frame with the straws was placed on the liquid nitrogen and the temperature monitored. When the temperature decreased to -140C the frame was manually turned over and the straws submerged in the liquid nitrogen.

The chart below shows the post thaw motility of straws frozen using both methods. In both cases the straws were taken directly from the liquid nitrogen and placed in a semen straw warmer set at 99F for one minute before being cut in the center.  A drop of the thawed extended semen was placed on a warmed slide with a warmed cover and then examined using a microscope for percent motility.

=>NOTE: Whenever motility was being evaluated at least two individuals separately estimate the percentage of mobile sperm.

Post Thaw Motility  - Comparison of Freezing Methods

Bull     Timed Lowering                                   Floating Rack Method – Height Above LN

               on rack into LN                       3cm above LN          4cm above LN                   5cm above LN

Sad             45%                                              50%                             45%                                 40%

Straw – 30M sperm/ml, Bullxcell

Sad              45%                                              45%                             25%                                  50%                                                                    Straw – 30M sperm/ml, Optidyl

Sad Eyes     40%                                               40%                             25%                                 30%

Straw – 45 M sperm/ml, Optidyl                                                               

#47                  45-50%                                       50%                            45%                                  25%                                                                Straw – 30 M sperm/ml Bullxcell   

#47                   35%                                              25%                             25%                                 25%

Straw – 30M sperm/ml  Optidyl                                                             

#47          30%          45M                                     20%                               40%                                                                                                      Straw - 45M sperm/ml  Bullxcell

#47              25%                                                    25%                                25%                                                                                                    Straw – 45M sperm/ml   Optidyl

Phantom            45%                                          45%                                   35%                                                                                                              Straw – 45M sperm/ml  Bullxcell

The following figure shows the temperature of the rack the extended semen straws were placed on during the two methods of freezing.  The gray, blue and red curves are for three freezing runs using the timed lowering of the rack through the LN vapor and into the LN and the yellow, light blie and green curves show the temperature change using the float and plunge method with the colors indicating how high above the LN the rack was floating until the temperature at that height decreased to -140C.

E.  Loading Frozen Semen Straws into LN Storage Tank for Preservation

Under SSARE Project FS21-335 after the straws were submerged in the LN at the end of the freezing process we would use tweezers and pick up the straws, place them in a Goblet which was already attached to a Cane and lower it into the LN tank. Every time a new straw was added the Cane would have to be raise out of the LN tank high enough to place the straw in the Goblet and then lowered again. Even being done in the cold room at 5C/41F the straws were going from -196C into a 5C environment long enough to place them in a Goblet and Cane and to then lower the Cane back into the LN tank at minus 196C. Furthermore, since each Goblet held five straws, the straws already in  Goblet were being raised up into 5C air and then back into minus 196 LN until the Goblet was filled with five straws. While this was done as quickly as possible, the straws were exposed to multiple temperature variations from minus 196C to 5C  for at least 8 seconds (The average time it took to fill a Goblet). The stress on the frozen extended sperm resulted in decreased overall motility and progressive motility.

The solution was simple:

• When freezing the straws, instead of filling the Styrofoam container with enough Liquid Nitrogen to immerse the straws, fill it a depth of 1.5-2 inches of Liquid Nitrogen.
• Once the straws are submerged on the rack in the Liquid Nitrogen at the end of freezing process, remove the rack leaving the straws on the bottom of the Styrofoam container.
• After several minutes select several straws for post thaw results. Remove them one at a time placing the straw in a semen warmer at 99F and move to the room with the microscope.
• After one minute in the warmer, cut the straw in the middle and place a drop of the extended semen p on a warmed slide with a warmed slide cover placed over it.
• The thawed extended semen is the examined by at least two people (experienced in estimating motility) who estimate the motility and who do not share their result until each person has examined it.
• This should be done for each variation of semen frozen (eg. bull, extender type, concentration).
• After determining post thaw motilities, the straws in the Liquid Nitrogen in the container can be loaded in prelabeled Goblets making sure that the Goblet is entirely submerged while being filled using 16 inch tweezers. After the Goblet is filled it is left in the Liquid Nitrogen until all the needed Goblets are filled.
• After all the Goblets are filled, a Cane, which holds two Goblets, is submerged in the Liquid Nitrogen and filled with two Goblets. The entire process being done with the Cane and Goblets submerged.
• The loaded cane is then removed from the Liquid Nitrogen and placed in the semen storage tank as quickly as possible. Note: Since the Goblets are closed on one end, they will contain a small amount of Liquid Nitrogen with the straws which will help reduce any temperature change during the few seconds it takes to move the loaded Cane into the storage tank.

E. Insemination

The critical issue in the actual artificial insemination of yaks is knowing when to inseminate. During the FS21-335 Project and the beginning of this project when we artificially inseminated the angus at the University of Kentucky with frozen yak semen we followed a slightly modified a standard seven day CIDR protocol used for beef cattle.

In this modified protocol we eliminated the injection of GnRh at the time of CIDR insertion, left the CIDRs in for seven days and upon removal injected the yaks with 5ml of Lutalyse.  Approximately 72 hours after the removal of the CIDRs, the time used in the beef industry, we inseminated the yaks and followed with a second insemination at approximately 96 hours.

Yaks do not show many signs of estrus. However, during the insemination of each yak cow Dr. Anderson was able to assess if the cow was close to estrus by the “slickness” the vaginal wall, smell and ease of insertion of the insemination gun into the cervix. However, we did not know if they were in standing heat or if ovulation had occurred.   

The fact that we did not know exactly when they were in estrus, and more exactly if ovulation had occurred contributed significantly to poor and inconsistent breeding results. The other factor was the quality of the semen we were using.

The quality of the frozen semen was tested by using unfrozen extended semen on a group of ten yak cows.  With the unfrozen semen we had a conception rate of 40%.  While with frozen semen we have had conception rates between 0 and 25% using the same sequencing protocol. So inconsistent semen quality (% post thaw motility) was a key issue as well as timing.

When we inseminated the angus we had a 66% conception rate with frozen semen (with good post thaw motility) which was excellent. Of course the reason it worked so well is we were breeding angus for which the seven day protocol works very well. 

• So good quality semen inseminated at the correct time did provide good results.

This left two things to be resolved:

1. Improved and consistent percent post thaw motility – which we resolved in the first year of this grant by eliminating semen exposure to temperature changes from Collection (use of warmers on the collection tube) to how we got the frozen semen into the Storage tank – described previously.
2. Knowing when to inseminate

Dr. Anderson researched different sequencing protocols being used at the yak research centers in China and India and, based on their insemination results,  recommended we use a five day protocol developed for Bos Indicus which consists of giving 5ml of Lutalyse at the time of CIDR insertion and a second 5ml injection of Lutalyse when the CIDR is removed. GnRh would be given after an insemination if the yak did not give signs of being close to estrus.

=>NOTE: The purpose of the GnRh was to induce ovulation within twelve hours so it could be inseminated by remaining viable semen. This works for beef cattle and was assumed it might work for yak.

 To test the Bos Indicus Sequencing protocol we ran four trials in which we sequenced three yak cows following this system and kept them in a corral around which we had positioned four game cameras. Twenty-four hours after removing the CIDRs we brought a bull into the corral with the cows and turned the cameras on letting them run for 120 hours post CIDR removal. 

• In all four trials estrus, as determined by how the yak cows reacted to the bull’s mounting, ranged from 60 hours to 110 hours.
• Furthermore, there was no clustering of estrus occurring around any specific time(s) although the majority were between 70 and 100 hours.

We still did not know when a  given cow was in estrus  within the overall time range of 60-110 hours.

To solve this we took a friendly bull that was no longer consistently producing semen good enough for the project and Dr. Prater performed an epididymectomy, removing the epididymus which holds the sperm before entering the vas deferens. In a bull the epididymectomy is far easier on the bull and less invasive than a vasectomy. This provided us with a completely intact bull who was not fertile.

This bull would serve two purposes:

• It is known from beef cattle that having a bull present with cows helps stimulate and "cluster" estrus; and,
• The behavior of the yak cows to the mounting by the bull would signal when cows were in estrus. We could tell this by placing Estrotech patches made for this purpose just above the tails of the yaks.   These patches turn a bright red from the pressure and movement of being mounted by the bull.

Our first attempt to use the Bos Indicus sequencing system with the "teaser" bull and use of tail patches was done on March 13th, and 14th, 2024 after we had made the three successful semen collections with high post thaw motility results. Twenty-two yaks were sequenced along with a Jersey cow and half Jersey heifer.

Approximately 48 hours after CIRD removal one yak started showing signs of being near estrus as detected by her movements using the Cattle Manager System which will successfully predict estrus in beef cattle based on their movements. 

By 60 hours after CIDR removal the bulls in an adjacent pasture were all along the fence-line. The teaser bull had mounted the one yak who showed signs of estrus earlier according to Cattle Manager.  This was known by the patch having been rubbed red.

At 70 hours post CIDR removal 5 yaks had been in or were in estrus (red patches from the bull’s mounting). All of the yaks were bred except a small 400 pound three year old female. We were going to wait to see if she would come into estrus. The Jesey and half Jersey heifer were also bred but had exhibited no signs of estrus. 

At 96 hours post CIDR removal 12 more yaks had red tail patches signaling having been or being in estrus.   All the yaks who had not been in estrus before were bred at this time including the young small female which still showed no sign of estrus and a twenty year old cow who also stilllshowed no signs.

 [The Jersey and half Jersey also showed no sign of being in heat by internal examination which would be expected since this was a Bos Indicus protocol and they would respond to the seven day Bos Taurus protocol.]

 Dr. Anderson, based on how the remaining two yak cows which had not yet come into estrus felt when inseminating them, estimated they would come into estrus in at about 108 hours post CIDR removal – ten hours after being inseminated at 96 hours post CIDR removal.

=>Note: Two ½ ml straws were used at each insemination. All of the straws contained a minimum of 30M extended sperm and the lowest post thaw motility was 30% so at least 9 million motile sperm per insemination.

 =>Note: Using the information from the Computer Assisted Semen Analyser the Progressive Motility of the post thawed extended semen was between 15% and 23% for the straws tested. Assuming two ½ ml straws with concentration of 30M sperm/ml and a progressive motility of 15% result in approximately 4.5 million or more progressively motile sperm at the time of insemination.

The twenty-two artificially inseminated yaks, the Jersey cow and half Jersey heifer were pregnancy tested on April 18, 204 using ultrasound:

                                  8 yaks initially conceived:          36% conception rate

                                  2 aborted post conception

                                  Jersey & half-Jersey heifer:        50% conception rate

The remaining open yaks were rebred April 20 and 21 after being given Lutalyse on the 18^th to bring them into estrus for a second round of artificial insemination.

                                       16 yak cows rebred

                                       Half jersey heifer rebred

Due to the heat of the summer it was decided to check pregnancy used Estrotech patches with a teaser bull present and not ultrasound because the stress of the heat combined with that of the ultrasound could induce an abortion.          

                                                4 yaks conceived second round of breeding     (4/18)= 22% conception rate

                                                Half – Jersey never came into estrus

Overall conception rate for the March-April AI: 12 conceptions out of 22 yaks = 54%, non yaks – 50%

=>The much-improved conception rate as compared to those of the first project FS1-335 can be attributed to improvements in processing the semen resulting in consistently better post thaw motility, using the Bos Indicus sequencing protocol, and having a better estimate f estrus through use of the Estrotech patches and the teaser bull whose presence might also have caused clustering of estrus in some of the yak cows.

We ran a second test of the Bos Indicus Artificial Insemination Protocol utilizing the Estrotech patches and teaser bull to better identify when the yaks were in estrus. The first insemination was done March 3, 2025. Eleven yak cows and a Jersey cow and half Jersey heifer were inseminated. Five of the yak cows were inseminated with semen collected from the bull Helios: A new bull we hadn’t collected from before. The remaining six yaks and the Jersey cow were inseminated using semen collected from a bull (Phantom) we had used previously and whose extended semen had consistently good post thaw motility and had resulted in previous AI pregnancies.

Of the five yaks inseminated using the semen collected from Helios:  0 conceptions      (0%)

Of the six yaks inseminated with the semen from Phantom:  3 conceptions         (50%)

Of the two non-yaks inseminated with Phantom’s semen:      1  conception        (50%)

There were two early abortions post conception.

The Computer Assisted Semen Analyzer POST THAW results done using frozen semen samples for Helios and Phantom taken from the processed straws in the March and April inseminations were:

                                                                Extender used: BullxCell

Bull                Total Motility at thaw     Progressive Motility at thaw      Progressive Motility at 3hrs  

Helios                         21.65%                             9.95%                                              5.35%   = 2.4M sperm                     

Phantom                   40.29%                             15.43%                                            20.26% =  9.1M sperm

[Total sperm contained per 0.5 ml straw: 22.5M                                     Two straws per insemination.]

Unfortunately we did not have this information for his March-April round of insemination. If we had, we would never have used the extended semen from Helios and used the BullxCell from Phantom or Sad: Both of whom had much higher progressive motility at 0 and 3 hours post thaw.

For the April insemination after determining which yaks were open or pregnant from the March 3-4 insemination by ultrasound we gave all the open yaks except two 5 ml of Lutalyse as the yaks were already synched from the Bos Indicus protocol used for the March 3^rd- insemination. The two not given Lutalyse showed no Corpus Luteum and as such the Lutalyse would have been ineffective.

Of those given Lutalyse (had Estrotech patches affixed with the teaser bull present):

• One yak showed estrus by Estrotech patch and teaser bull behavior at 5pm, April 13^th – so she was inseminated more than twelve hours after showing estrus;
• Six of the remaining eight yaks were showing good signs of estrus based on the Estrotech patch and teaser bull on the morning of the 14^th ; and,
• Two did not start to show any signs of estrus until the morning of April 15^th.

The result was

• 1 yak inseminated more than 12 hour post estrus (but this would be during ovulation)
• 6 yaks in estrus (as indicated by Estrotech patch and teaser bull) inseminated April 14-15:
• 2 yaks just starting to how signs of estrus on the morning of April 15^th and inseminated before full signs of estrus.

Dr. Anderson, the Reproduction Specialist, could tell by examination that the first yak was post estrus and said the six by internal examination were in estrus. These seven yaks would have had the greatest chance of conception.

=>The two yaks who were just starting to show signs of being mounted according to their Estrotech patches and their unwillingness to stand for the teaser bull probably would not have conceived since according to the CASA analyses of the semen three hours seems to be the approximate limit of having sufficient progressively motile sperm to inseminate (although the sperm would survive longer in the reproductive track than in a test tube at 99F.)

Our belief is that the insemination protocol [Bos indicus, Estrotech patches, teaser bull first round followed by Lutalyse, Estrotech patches and teaser bull] worked very well in telling us who was really ready to breed. The problem, as noted, was the semen.

=>Even though this project ends September 30, 2025, we have scheduled another round of insemination first week of December, 2025 wit the second round done approximately thirty days later. This time we will use semen from Phantom and the Bos Indicus – Estrotech patches-teaser bull protocol. The results from this round of insemination will be attached to this report.

1. Research on The Hybrid Yaks

The plan of the project was to obtain information preliminary pieces of data concerning yak x angus hybrids.

1. Artificial insemination pregnancy rate using extended yak semen and the standard beef AI seven-day protocol;
2. Weight gain information: How weight gain of yak x angus hybrids compared to that of yaks; and,
3. Quality of meat in terms of fat content and protein of grass-fed yak x angus as compared to grass-fed yak.

=>As noted previously, six University of Kentucky angus cows were artificially inseminated with extended yak semen with known good post thaw motility.  Four of the six, 66%, became pregnant and calved in the spring of 2024 with birthweights between 50 and 60 pounds. Yak birth weights run between 25 and 40 pounds.

Hybrids at approximately one month.

=>A fifteen week comparative weight gain study on typical eastern Kentucky mixed pasture grass which was approximately two-thirds fescue endophyte infected fescue was held May 3^rd to August 15^th, 2025. The results were as follows:

                                                                                 Weight              Weight                   % Increase

Hybrids                                          May 2, 2025        August 22, 2025        in Body Weight           Daily Gain

M201                       F                              572                   738                                29%                         1.49lb

M202                       B                              631                     775                                23%                        1.3lb

M203                       F                              464                    630                               36%                         1.49lb

M204                       B                              570                     700                               23%                         1.18lb

Yaks

Yak AB                     B                              462                           535                       16%                             .66lb

Yak MB                    B                              458                          535                      17%                             .69lb

Yak CB                     B                              453                          503                       11%                            .45lb

Yak TB                     B                              389                          453                       16%                            .58lb              

                           Hybrid heifer at 15 months                                                                      Hybrid bull at fifteen months      

NOTE: We were not be able to do the meat analysis based on one of the hybrid bulls within the time frame of this grant. Once one of the hybrid bulls is ready to process for meat, a sample will be taken and analyzed and the results included as an addendum to this report.