ANS 3319C Reproductive Physiology & Endocrinology Lab
Semen Evaluation & Cryopreservation: Bovine & Equine
Objectives
1)
2)
3)
To introduce the components of semen evaluation used in preparing semen for freezing
(cryopreservation) using the beef bull as our model.
To provide “hands-on” experience in performing the steps of a semen evaluation.
To provide “hands-on” experience in the process of cryopreservation of equine semen.
Evaluation of Bovine Semen for Cryopreservation
Visual Examination of Semen Sample
The gross appearance of freshly collected semen is usually the first measure of semen quality.
Neat (unaltered) semen appears as a thick whitish to slightly yellowish fluid. The thickness of
the semen sample is a reflection of the number of sperm present. There should be no odor
associated with the semen sample. Potential odors are suggestive of an infection or the
presence of urine, which could be detrimental to fertility of the sample. Other problems
considered to be detrimental can also be detected in the color of the semen such as blood,
urine, and feces, which cause the semen to be pink to brownish. White clumps or flakes indicate
pus and the presence of an infection in the reproductive tract of the male. Sometimes debris
from the semen collection site might also be found in the semen sample such as sand, dirt, straw
or other bedding material. Ejaculates that are abnormal in color or appearance should be discarded at this point in the processing.
Viability Components
1. Motility
Motility of semen is one of the simplest viability characteristics to evaluate. Semen is diluted
with an isotonic buffer and then examined either under a bright field microscope or a phase
contrast microscope using a heated stage (35 - 39°C). Slides and coverslips are prewarmed on a slide warmer prior to adding warm semen (35 - 39°C). Several fields of view
are examined and the percentage of motile cells estimated to the nearest 5 or 10%.
Although it is important to look for progressively motile sperm (sperm moving in a nearly
straight line) it may be just as relevant in evaluating viability to determine if sperm are just
motile at all (total motility, sperm being able to propel themselves with a beating tail). For the
first time evaluators, the process of estimating percentages of motile sperm often appears
difficult and inaccurate. Critical in this is the standardization of the procedures for diluting the
semen and making the slides. During the observations on the microscope it is also important
to use the fine focus and continually focus on sperm in different planes. Semen samples kept for processing should have greater than 60% motile sperm.
•
Gross Motility- gross swirling pattern of undiluted semen looks like a large school of fish.
•
Individual Motility
◊ An estimate of individual sperm movement. Sperm should move progressively from
one point to another in a relatively straight line.
◊ Dilute raw semen with physiological saline (0.9% NaCl).
◊ Count ten sperm cells in ten different areas of the slide. The number of sperm that
are progressively moving will be the estimate of percent motile.
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
2
2. Morphology
The structure, or morphology, of sperm cells has been studied extensively using light and
electron microscopy techniques. The sperm is a highly structured cell that is stream-lined to
deliver DNA to the oocyte. To achieve this goal, the DNA is highly condensed and packaged
on the nuclear matrix in a unique and specific species manner. Alterations in the packaging
or DNA content of sperm results in changes in the morphology of the sperm head. The tail of
the sperm contains the locomotion apparatus and without its proper function can not deliver
the DNA in the sperm head to the oocyte.
Early experiments observed that semen from subfertile and sterile bulls contained increased
amounts of abnormally shaped sperm. This phenomenon has been observed in other
species as well. It appears that abnormalities to the sperm can cause decreased ability to
reach the site of fertilization, inability to fertilize oocytes, or inability to sustain embryonic
development. While it is important to monitor sperm morphology, it is particularly important
in new males entering the production system. More established males are evaluated less
frequently but still need to be done on a routine basis.
There are several classification schemes for morphological characteristics in the bovine.
While some are based on the step where the abnormality occurred such as during
spermatogenesis (primary abnormalities) or epididymal maturation (secondary abnormalities)
while others are based on the severity of the abnormality in relationship to fertility. The most
common method is one based on the structural component of the sperm involved.
Abnormalities associated with the head are classified as primary and those associated with
the mid-piece or tail classified as secondary. Some researchers separate the midpiece and
tail abnormalities into secondary and tertiary, respectively. Primary abnormalities are
believed to have more of an impact on fertility than secondary abnormalities. Some of the
primary and secondary abnormalities of bovine sperm are presented in Figures 1 and 2.
Ejaculates should have greater than 70% normal sperm with no more than 20% sperm with primary abnormalities.
3. Acrosome integrity
The acrosome is a secretory vesicle that is a sac-like structure below the plasma membrane
and covering the anterior nucleus of the sperm head. The integrity of the acrosome is very
closely associated with sperm viability because damage to the plasma membrane can trigger
a disintegration of the acrosome that can be observed on a phase contrast or differential
interference contrast (DIC) microscope. While unfixed sperm can be examined to determine
the percentage of sperm with intact acrosomes, generally sperm are fixed with glutaraldhyde
or other fixatives before examination. Semen samples with less than 70% sperm with intact acrosomes should be discarded before processing.
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
Figure 1. Morphology of bull spermatozoa – primary abnormalities of the head. Normal
Decapitated
Macrocephalic
Microcephalic and
Stump Tail
Round head
Pyriform
Pyriform
Tapered
Ruffled acrosome
Round and Double
Tail
Microcephalic
Craters (diadem)
3
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
Figure 2. Morphology of bull spermatozoa – secondary abnormalities of the tail. Proximal
Cytoplasmic
(Protoplasmic)
Droplet
Translocating
Cytoplasmic Droplets
Tail Opening
following Droplet
Translocation
Tail Opening
following Droplet
Translocation
4
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
5
Figure 2. (Cont.) A Coiled tail with droplet; B Coiled double tail; C "DAG" defect; D Folded tail; E Filamentous; F Double tails; G Corkscrew midpiece with droplet; H Corkscrew midpiece 4. Live-Dead
In addition to the viability measures such as motility and acrosome integrity it is also
possible to determine the percentage of live sperm using a differential vital stain. Early
staining procedures used histological stains such as eosin that would only penetrate
sperm with a compromised plasma membrane (Figure 3). Sperm were then dried on a
slide and counted as viable (unstained) or dead (stained). Other procedures use a
combination of fluorescent dyes such as sybr-14 which stains viable sperm green and
propidium iodide which stains dead sperm red (Figure 3: Please view this figure on the
web page to see the colors). Semen samples should have more than 70% viable sperm by a vital stain assay prior to processing.
•
Classification of viability.
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
◊
◊
◊
◊
6
Use a live-dead smear to arrest motility & assess the percent of live/dead sperm.
Based on the premise that as sperm die, their membranes degenerate.
Percent live/dead sperm - identifies the percentage of sperm that have intact
membranes, which is indicative of the number of live sperm in a semen sample.
An eosin/nigrosin stain is commonly used for this purpose.
A
B
C
D
Figure 3. Illustration of sperm
which have been treated with the
dual-staining technique. A) Live
sperm with intact acrosome. B)
Live sperm without acrosome
(true acrosome reacted). C)
Dead sperm with intact
acrosome. D) Dead sperm
without acrosome (false
acrosome reacted). Picture
below using fluorescent dyes to
mark viable (green) and dead
sperm (dead).
5. Concentration
The accurately determine the number of sperm per unit volume is a central job of
freezing semen. Concentration is usually determined using specialized equipment
containing a spectrophotometer. The concentration of sperm in the neat semen is
needed to determine how to dilute semen and provide adequate sperm in each breeding
dose. The number of sperm ejaculated varies greatly among males but is dependent on
age, size of the testes, and the efficiency of spermatogenesis/gram of tissue. The sperm
production on any day is also dependent on the collection frequency and the intensity of
the sexual stimulation employed. While young bulls produce from 2 to 5 ml of semen,
older mature bulls might produce 6 to 15 ml. The concentration of sperm in neat semen
is 500 - 2,000 million sperm/ml. Semen that is below 500 million sperm/ml will not yield large numbers of insemination doses and might be considered a poor choice to continue on in processing.
Semen used for cryopreservation is more rigorously evaluated than semen collected for a
breeding soundness exam in all evaluation categories. Since sperm cells need to survive
both the freezing and thawing process, starting with the highest quality semen is essential.
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
7
Extension and Freezing of Bovine Semen
Semen collected from farm animals and used for AI is either extended and used “fresh” within
several days of collection or extended and frozen for use at a later date. The primary purpose of
extending semen is to maximize the number of females that can be inseminated with a single
collection. Furthermore, extending semen also provides nutrients for the sperm and creates a
physiologically safe environment for sperm to survive so fertility is maximized. For horses and
pigs, the majority of inseminations are conducted using fresh extended semen. In contrast, in
the bull and ram it is not practical to use fresh semen so the semen is extended, packaged into
small straws, frozen, and stored in liquid nitrogen for future use. Depending on the species and
volume of semen collected during an ejaculate, the number of inseminations attained after
extension varies from 1 - 4 in the stallion, 5 - 15 in the boar, and 100 - 500 in the bull.
A. Characteristics of Semen Extenders
1) Functions of extenders.
♦ Provides nutrients as a source of energy.
♦ Protects against the harmful effects of cooling and freezing, which take place in liquid
nitrogen (-196°C).
♦ Provides buffers to prevent harmful shifts in pH.
♦ Maintenance of proper osmotic and electrolyte pressure.
♦ Increase semen volume so it can be used for multiple inseminations.
♦ Extenders must provide an isotonic environment; hypertonic solutions dehydrate
sperm while hypotonic solutions cause sperm to swell and rupture.
2) Components of extenders.
♦ Buffers - Function to control pH 6.7 to 7.0. Sodium citrate, egg yolk, tris buffers are
commonly used.
♦ Lipids - Provides protection of sperm membranes from temperature changes. Skim
milk and egg yolks are good sources of lipids.
♦ Nutrients - Provide energy for sperm. Fructose and glucose are typically used.
♦ Antibiotics - Prevent bacterial growth.
♦ Glycerol - Is a cryoprotective agent for freezing semen. It protects against the lethal
effects of freezing to prevent crystallization of water within the sperm cells, which
eventually allows sperm cells to be frozen rapidly. Formation of ice crystals results in
puncture of cell membranes resulting in the decrease in membrane integrity.
3) Example Extender: egg-yolk and glycerol
♦ Egg yolk - Citrate (Fraction A)
20% (vol/vol) Egg yolk
80% (vol/vol) of a 2.9% (weight/vol) Na Citrate dihydrate
1000 IU Penicillin/mL
1000 μg Streptomycin/mL
♦ Egg yolk - Citrate + Glycerol (Fraction B)
20% (vol/vol) Egg yolk
66% (vol/vol) of a 2.9% (weight/vol) Na Citrate dihydrate
14% (vol/vol) glycerol - provides final concentration of 7% glycerol
♦ Glycerol is usually added after the semen has been cooled to 5°C. This prevents
morphological damage to sperm compared with glycerol added at room temperature.
The glycerol fraction is typically added in three equally timed steps and volumes. The
final volume of extender will contain equal amounts of Fractions A & B.
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
B. Calculations for Processing Semen for Freezing
1) Determine the number of motile sperm per dose of semen to be inseminated. Some bull
studs will actually adjust the number of sperm cells frozen based on percent motility and
(or) abnormalities of the collection and include more sperm cells per dose.
♦ Target concentration to freeze: 20 × 106 live, motile sperm per dose. 2) Not all sperm cells survive the freezing/thawing process. Many of the cells die because
of damaged cell membranes (10 to 40 percent of sperm). Therefore, more sperm cells
are frozen than are needed.
♦ Target concentration for insemination: 14 ‐ 15 × 106 live, motile sperm per dose. C. Example Calculations for Extending Bull Semen:
Semen Volume:
7 mL
Concentration:
1.6 billion/mL
Total sperm in ejaculate:
11.2 billion
7 ml semen × (1600 × 106/mL) = 11,200 × 106 total sperm in collection
Total sperm per breeding unit desired: 20 million motile sperm per 0.5 mL straw
Total number of breeding units:
(11,200 × 106 total motile sperm) ÷ (20 × 106 total motile sperm/dose) = 560 doses
Total volume of extender needed:
560 × 0.5 ml/straw = 280 ml extender needed
140 mL fraction A; 140 mL fraction B
(Contents of fractions A and B were described on the previous page)
D. Freezing Bull Semen
1) Semen is collected using an artificial vagina and the collected sample is placed in a dryblock or water bath at 90 to 95°F (32 to 35°C) during the evaluation period.
a) Qualitative (morphology, motility) and quantitative (concentration) measurements are
made to allow determination of proper volumes for extending semen.
b) Extension rates may be adjusted based on the qualitative and/or quantitative
measurements to ensure that the target concentration at insemination (14 - 15 x 106
live, motile sperm/dose) is achieved.
c) Antibiotics are added to the neat semen and it is allowed to sit 3 to 5 minutes.
2) Semen is extended for freezing
a) Combine 3 parts of Fraction A with 1 part semen at 35°C.
8
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
9
b) Cool Fraction A + semen to 5°C in a cold room. This process typically occurs over 2
hour so sperm are not cold shocked. Add remainder of Fraction A to final volume.
c) Add Fraction B (contains glycerol) to extended semen at 5°C.
♦ The glycerol fraction is usually added in three equal steps, 15 minutes apart.
d) Packaging
♦ Today, most bull semen is frozen and stored in either 0.5 or 0.25 mL polyvinyl
straws. The ends of the straws are sealed after the semen is loaded to prevent
liquid nitrogen from entering the straw.
♦ Straws are labeled to identify the semen it contains including items like sire name,
appropriate breed code, breed association registration number, date frozen, and
processing location.
e) Straws are allowed to equilibrate in a cold room (5°C) for 4 to 6 hours to allow for
morphological changes that occur to the sperm cell membranes, which increases the
sperm cells chance of survival during the freeze-thaw process.
f)
Straws are placed in a horizontal freezing rack and placed approximately 3 cm above
the surface of the liquid nitrogen.
♦ Freezing is usually accomplished in 7 to 10 minutes in the liquid nitrogen vapor
after the semen reaches -100°C.
g) Once the temperature of the semen reaches -100°C, the semen is plunged into liquid
nitrogen (-196°C).
h) Packaged semen is stored in liquid nitrogen (-196°C) where it can be stored for years
while still maintaining sperm viability.
3) Post Thaw Semen Evaluation
a) After semen has been frozen, a post-thaw examination will be conducted to
determine how well the semen endured the freezing/thawing process. A straw of
semen is thawed in a 37°C water bath for approximately 30 to 45 seconds.
b) Samples are evaluated for motility, morphology, and viability as previously described.
c) Additional test could be conducted including a stress test where the semen is
incubated in a water bath at 37°C for approximately 2 to 3 hours and acrosomal
integrity will be evaluated.
d) Semen with poor motility and (or) decreased viability will typically not be released for
use or sale. Post thaw motility rates are typically between 50 to 70%. As motility
decreases, subsequent pregnancy rates can also decrease.
4) There is considerable variation in how semen freezes between individual bulls as well as
day-to-day variation between collections. Therefore, excellent quality control programs
must be maintained in order to consistently produce quality frozen semen.
For reference purposes, parts of this handout have been adapted from: 1) Dr. John Parish’s web site University of Wisconsin
(http://www.wisc.edu/ansci_repro/), Department of Animal Science for his Animal Sciences Reproductive Physiology class (2003). 2)
The Artificial Insemination and Embryo Transfer of Dairy and Beef Cattle, H.A. Herman, J.R. Mitchell and G.A. Doak, Interstate
Publishers, Inc., (1994)
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
10
Utilization of Cooled and Frozen Equine Semen
The continuing expansion of assisted breeding techniques like AI offers farm managers multiple
options for allocation of stallion resources. Relative to stallion management, processing semen
for shipment and (or) artificial insemination appears to be the most emphasized of all the
assisted breeding techniques at this time. Commercially available methods for semen transport
include the use of cooled or frozen semen. Each of these methods offers advantages and
disadvantages for on-farm use. We have previously described (see Equine Semen Collection
and AI lab) the advantages and disadvantages of cooled semen, but let’s review them again
along with advantages and disadvantages of frozen semen.
1. Advantages of Cooled Shipped Semen include the elimination of costs that pertain to
shipment of mares to breeding facilities that are long distances from an owner's farm,
reduction of stress and disease transmission to mare and foal, the genetic pool is increased,
superior genetics may be promoted through the use of semen from different parts of the
country/world, mare care costs will be reduced, and it is easily incorporated into most current
farm management schemes.
2. Disadvantages of Cooled Shipped Semen include packaging and shipment costs,
increased costs in mare management, semen from some stallions may not be suitable for
cooling, and personnel receiving the semen may lack the technical skills to properly handle
the semen.
3. Advantages of Frozen Semen include the elimination of certain costs pertaining to horse
shipment, reduction of stress and disease transmission to mare and foal, the breeding
season is not delayed due to performance events, semen from valuable stallions may be
stored and utilized at a later date helping to increase the genetic pool even after the stallion
has died, genetic pool may be increased, and semen may be collected, frozen, and stallion
gelded thus preserving the breeding capability without the problems associated with an intact
male.
4. Disadvantages of Frozen Semen include the considerable technology and skill required to
successfully freeze semen, considerable cost and intensive management are needed to
prepare for frozen semen insemination, genetic saturation of a particular stallion may occur if
breedings are not limited, and the most serious disadvantage is reduced pregnancy rates
using frozen/thawed semen.
In deciding what shipment techniques should be utilized in a stallion's management as well
as a farms management program, many questions should be answered before a facility
decides to commit and expend resources/money to establish one or both of these shipping
capabilities. Questions that should be carefully considered include:
♦ Overall objectives and management plan for implementing a shipment program
♦ Amount of semen shipped from farm. And is frozen semen really applicable for farm?
♦ Does or will the farm ship domestically, internationally, or both? International shipments
must be prepared to meet USDA-APHIS requirements, which may include major
modifications to existing structures and laboratories.
♦ Are stallions on the farm utilized for performance or show events that interfere with
normal breeding and training of the stallion during the spring breeding season, which
may facilitate freezing semen on certain sires.
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
11
Extension and Freezing of Equine Semen
1. Extending Ejaculated Semen
In preparation for freezing equine semen, the semen is extended in a manner similar to that
described for insemination of fresh semen. The ejaculate will be evaluated for volume, motility,
and concentration as described previously in the stallion semen collection lab. The semen will
be extended to obtain a final concentration of 50 x 106 sperm / mL at which time it is ready to
process for the freezing process.
2. Processing Semen for Freezing
Compared to beef cattle, process of freezing equine semen is straight forward and easy to
conduct on farm. The freezing media is typically purchased as a single frozen package that is
thawed immediately before use. It contains egg yolk, lactose, glucose, sodium citrate dehydrate,
disodium EDTA, sodium bicarbonate, glycerol, and an antibiotic (Ticarcillin). The process does
not require semen to be cooled before freezing as indicated in the bull. The process to be used
in lab is outlined on the last page of this handout.
3. Packaging Semen for Freezing
The standard package for frozen semen in the cattle industry is the 0.5 cc straw. With
standardized freezing procedures, a large number of units are frozen and inseminated each year
in the beef cattle industry, which is not yet the case in the equine industry.
The 0.5 cc straw has a number of advantages including familiarity, access to related products,
and good geometry for freezing and thawing. The main disadvantage is its small size and the
amount of semen it can hold. The 0.5 cc straw was designed to deliver a breeding dose of
semen in cattle of approximately 20 × 106 sperm cells. In contrast, horses require 10-20 times
more sperm in a breeding dose of approximately 800 × 106 sperm cells. Consequently, multiple
0.5 cc straws must be used to make up a single insemination dose. Therefore, approximately
6
200 × 10 sperm cells are frozen in a 0.5 cc straw and four single dose straws of semen
6
make up a breeding dose, which contains approximately 800 × 10 sperm cells. Even
though having to use multiple 0.5 cc straws to make up a breeding dose, the good freeze/thaw
characteristics available with the 0.5 cc package argue for its continued use in the equine.
4. Storage of Frozen Semen
Regardless of how semen is frozen or packaged, frozen/packaged semen is stored in liquid
nitrogen (-196°C), which is stored in vacuum lined tank. Liquid nitrogen tanks are capable of
holding liquid nitrogen for an extended time period and maintaining the temperature of -196°C.
5. Thawing Frozen Semen
Thawing instructions are typically different for each package type and laboratories that freeze
and packaged the semen. Packages impermeable to water will usually be thawed in a water
bath. For all semen handling procedures, direct contact between water and semen should be
avoided since water is toxic to sperm cells. When semen has been thawed in a water bath, the
package should be wiped dry before opening. For 0.5cc straws the thawing process is a single
step process whereby semen is placed in a 37°C water bath for 30 seconds. It is also important
to do a post thaw motility exam on each ejaculate of semen frozen to determine how well the
sperm survived the freezing/thawing process. For equine semen, post thaw motility rates are
low and typically around 30% motility. Semen that has < 30% post thaw motility rates should
probably not be used in an insemination program. In cattle, post thaw motility rates are
considerably greater and are typically 50 to 70%.
ANS 3319C Reproductive Physiology & Endocrinology Lab: Semen Evaluation and Cryopreservation
12
Equine Semen Freezing and Thawing Protocols
a. Semen Collection and Evaluation: Collect semen from stallion and pour ejaculate into a warmed
graduated cylinder and record total volume. Evaluate semen for motility and concentration and record
the numbers. The semen can also be evaluated for morphology as described for the bovine.
b. Extending Semen: Determine the amount of pre-warmed EZ-Mixin BF (no antibiotic) extender to add
to the ejaculate to obtain a final extended concentration of 50 x 106 sperm / mL.
Initial volume of raw semen (mL) × concentration (×106) = total sperm in ejaculate (×106)
Total sperm in ejaculate (× 106) ÷ 50 × 106 sperm/mL = Total volume
Total volume (mL) – semen volume = volume (mL) of extender needed
c.
Add 40 mL of diluted semen from step (b) to 50 mL centrifuge tubes.
d. Centrifugation to Concentrate Sperm: Centrifuge tubes for 13 minutes at 400 x g.
e. While tubes are being centrifuged, fill styrofoam freezing container to first line with liquid nitrogen.
Temperature inside the container will be approximately -165°C (+/- 5 °C).
f.
Isolation of Sperm Pellet: : After centrifugation, aspirate off the supernatant leaving a 0.5 mL pellet in
bottom. This pellet contains the sperm cells.
Total number of sperm in each centrifuge tube
(40 mL tube) (50 × 106 sperm/mL) = 2000 × 106 sperm/tube
(2000 × 106 sperm/tube) (.80*) = 1600 × 106 sperm/tube
*(Allows for only 80% return of sperm after centrifugation & removal of supernatant)
g. Addition of Freezing Extender: Add 3.5 mL of freezing extender (Ez-Freezin-LE) to each tube, mix
(1600 × 106 sperm/tube) ÷ (400 × 106 sperm/mL) = 4 mL total volume
(4 mL total volume) - (0.5 mL sperm pellet) = 3.5 mL freezing extender to add to pellet
If you have centrifuged partial tubes, calculate how much freezing extender to add to pellet
_________mL in centrifuge tube ÷ 40 ml = _________ × 3.5 = ___________mL to add to pellet
h. Combine all of the tubes into one tube and mix
i.
Packaging Semen for Freezing: Load 0.5 straws with semen, bubble end of straws with bubbling
comb, place straws into PVC powder, and dip end of straw with PVC powder into water to seal. Label
straws as needed.
j.
Freezing Process: Place straws on freezing rack and place rack in bottom of stryofoam container.
The rack will sit in the vapor above the liquid nitrogen. Allow liquid nitrogen temperature inside
freezing container to return to -120°C, at which time straws can be plunged into the liquid nitrogen (196°C).
k.
Storage: Transfer straws to cooled goblets/canes for storage in liquid nitrogen (-196°C) until needed.
l.
Post Thaw Motility Test: Thaw straws for 30 seconds in a 37°C water bath. Extend in 2.5 mL of EZ
Mixin BF. Evaluate motility at 0, 30, and 60 minutes post thaw.
m. Processing Frozen Semen for AI Thaw 4 straws (1 breeding dose) as previously described.
Combine straws and dilute in 10 mL of EX Mixin BF. Inseminate mare.