ABDULLAH Cooled and Frozen Semen Technology Loomis - semen.pdf · Cooled and Frozen Semen Technology Quality Control and the Impact on the Breeding Industry Paul Loomis ABDULLAH 1984

Cooled and Frozen Semen Technology

Quality Control and the Impact on the Breeding Industry

Paul Loomis

ABDULLAH

1984 Olympics Team Gold & Individual Silver

Principles of Cryopreservation and Theory of Cell Damage

Requirements for Fertilization

• Motility (progressive and then hyperactive)• Normal functional morphology

– Intact membranes until encountering oocyte• Survival in female reproductive tract

– Proper interaction with oviductal epithelial cells and secretions

– Must be at the right stage of maturation at the right time in the right place

System Design

• Billions of spermatozoa deposited in natural mating

• Female reproductive tract acts as filter for “normal” spermatozoa

• Thousands survive and make it to oviduct• Ensures that several are available and at

the correct stage of development when oocyte ovulated

Fig. 38. Conceptualization of spermatozoon–oocyte interactions. (A) Spermatozoon negotiates passage through the intercellularmatrix of the cumulus oophorus. (B) Initial adhesion of the equatorial region of the spermatozoon with the zona pellucida. (C) Inductionof the acrosome reaction. (D) Spermatozoal penetration of the zona pellucida at an oblique angle, with entry into the perivitelline space.(E) fusion of the post-acrosomal spermatozoal membrane with the microvillar region of the oolemma. (F) Establishment of a gatewayfor spermatozoal nucleus entry into the cytoplasm of the oocyte, as well as entry of spermatozoon cytosolic molecules (presumablyspermatozoon-specific phospholipase C) that serve as a signaling mechanism for activation of the oocyte and exocytosis of oocytecortical granules. Modified from Primakoff et al. 2002 and Rubinstein et al. 2006.760,789

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Non compensable

Performing a Test Freeze

Collection EvaluationPreparation•Volume•Concentration•Motility•Morphology slides

Processing

DilutionCentrifugation1st ExtensionHemacytometerFinal extensionPost-cent motility

Packaging

Freezing Storage Post-thaw Evaluation

Initial Evaluation

Measure and record

Measure and record

Volume

ConcentrationAccuracy of Densimeter and Spermacueat extreme concentration ranges

Initial dilution madeat this stage

Motility Evaluation Calculate TSPrepare morphologyslide

Initial Dilution & Centrifugation

• Variables– Extender used

• How to determine?– Dilution Rate – Centrifugation time and speed

Centrifugation of Semen

• Conventional 300 – 600 x g 10-15 min• Cushioned 1000 x g 20-25 min

• Recovery conventional 70 – 80%• Recovery cushioned 90 – 95%

• Potentially less damaging to sperm



500 µl Eqcellsire

Semen Extended 1:1 with SMG

Following centrifugation22 minutes 1000 x g

Extender and seminal plasma

Sperm layer

Eqcellsire

1. Aspirate Supernatant

2. Use fine tip disposableplastic pipet to carefullyremove Eqcellsire

Factors Affecting Recovery

• Sperm left in supernatant• Sperm lost when removing cushion



Figure 2. Plastic 15-mL conical tube, demonstrating a sperm pellet at bottom of tube following centrifugation in EquiPure solution. For this procedure, the centrifugation tube was loaded with 2mL of EquiPure bottom layer, followed by 2 mL of EquiPure top layer, and 1 mL of extended semen containing approximately 500 x 106 sperm. The sperm pellet at the bottom of the tube contains the sperm population most free of morphologic defects, whereas the thin white line at the interface of the bottom and top layers of EquiPure contains sperm more likely to possess morphologic defects. The thick white line overlying the clear EquiPure layers contains extender, as well as somatic cells and premature germ cells. This layer may also contain sperm with pronounced morphologic defects that would result in markedly reduced buoyancy.

From Varner, et al, J.Equine Vet Sci, Vol 28, No 11 (2008)

High % morphologically normal sperm

EquiPure bottom layer

Abnormal Sperm

EquiPure top layer

Extender, other cells

EquiPure for Sperm Separation

Following centrifugation 30 min at 200 x g

Initial resuspension

• Pellet quality• Seminal plasma remaining• Pellet resuspension• Extender addition

– Calculation of resuspension volume– Temperature

Calculating Extended Concentration

Total Sperm X 0.8 (estimated recovery) = Volume of initialDesired initial concentration resuspension

Variables

• % normal morphology from last depletion• initial motility• previous post-thaw motility• previous fertility

>60 % = 200 M/ml50-60% = 225 M/ml<50% = 250 M/ml

Final Extension

NucleoCounter count X initial resuspension volume = final resuspensionDesired final concentration volume

Perform post-centrifugation motility

Load Straws

• Flush straw filler (Minitube)– alcohol – water – extender

• Countersink sealing balls• Change bushings frequently• Change fill tubing for eachfreeze



Controlled Rate Cell Freezers

Properly stored frozen semen can maintain viability for many

years.

Frozen Semen Storage

SBSW – Texas >55,000Doses Stored

SB

S-M

aryl

and

>35,

000

Dos

es S

tore

d

Post-Thaw Evaluation

• Thaw 2 straws, combine contents• Culture thawed semen for mare pathogens• Dilute in appropriate extender• Incubate 25 minutes• Perform post-thaw motility at 30 minutes

post-thaw– Incubated motility provides better correlation

with fertility

Minimum Quality Standards for Minimum Quality Standards for Commercial Frozen SemenCommercial Frozen Semen

•• Each dose of semen should containEach dose of semen should contain–– >>200 million progressively motile sperm200 million progressively motile sperm–– >> 30 % post30 % post--thaw progressive motilitythaw progressive motility

•• Each ejaculate cultured for bacterial Each ejaculate cultured for bacterial pathogenspathogens

•• All stallions tested for EVA, CEMAll stallions tested for EVA, CEM



Problem

• Many mare owners and veterinarians have little confidence in frozen semen quality

Why?

• Too much variability in quality of semen sold on the commercial market.

• Semen sold without guarantee of quality or fertility.

We should be able to assure breeders that a dose of semen

1. Contains a certain number of sperm2. Upon proper thawing will possess a

certain minimal motility3. Is free of potential pathogens or

significant contaminating bacteria

05

10152025303540

1 2 3 4 5 6 7 8 9 10 11 12 13

Ejaculate

Post

-thaw

pro

g. M

otili

ty

(%

) MeasuredExpected

Post-thaw Motility of 13 Ejaculates of Imported Semen

from a Single Stallion

All semen reported>35% post-thaw motility

0

100

200

300

400

500

600

1 2 3 4 5 6 7 8 9 10 11 12 13

Ejaculate

MeasuredExpected

Sperm Concentration of 13 Ejaculates of Imported Semen

from a Single Stallion

Spe

rm C

once

ntra

tion

Variation in Cooled Semen Quality

Shipment characteristicsShipment characteristicsn = 282 shipments from 44 stallionsn = 282 shipments from 44 stallions Mean Mean ++ s.d.s.d. RangeRange

Volume (ml)Volume (ml) 56 56 ++ 2020 20 20 -- 159159

Concentration (10Concentration (1066/ml)/ml) 43 43 ++ 2020 6 6 -- 200200

Progressive motility (%)Progressive motility (%) 44 44 ++ 1616 1 1 -- 8585

Morphologically normal (%)Morphologically normal (%) 51 51 ++ 1313 4 4 –– 8585

PMMN sperm/dose (10PMMN sperm/dose (1066)) 598 + 604598 + 604 8 8 -- 42574257From Burns, TE, 2000



Variability in Cooled Semen Doses

• 201 doses from 67 stallions• 36 different EU-approved centers

(33 Germany, 1 Belgium, 1 Netherlands, 1 Austria)

average rangeConcentration 68 M/ml 14 - 327Total Sperm 1.0 billion 0.2 – 5.2

Total Motility 83% 30 - 96% Morph. Defects 45% 17 - 87

Solution• Strict adherence to post-thaw quality

standards.• Objective analysis of semen quality• Fair and equitable marketing system for

frozen semen with guarantees of quality or fertility.

• Sell pregnancies……not doses of semen!!

Concentration

• Direct Counting– Hemacytometer– Flow cytometer– CASA– NucleoCounter

• Inferred Concentration– All photometers– Must be calibrated by direct counting

• Usually hemacytometer

Correct Hemacytometer Protocol

§ For accuracy a minimum of 4 chambers is required.

§ Variance associated with the technique is diminished by 30% whentwice as many chambers are counted, 50% with four times as many chambers and 60% with 6 times as many chambers (Lorton et al, 1984).

§ Mean differences in the order of 20% can be expected from duplicate determinations by the same technician (Freund and Carol, 1964).

§When two technicians count the same semen sample, 95% of the time the second count will be ± 52% of the first (Freund and Carol, 1964).

HEMACYTOMETER“Gold Standard” Only if done correctly

•Accurate pipeting, dilution & chamber loading•Humidity chamber•Standardized counting method•MINIMUM of 4 separate chambers counted

Photometers

• Advantages– Rapid– Inferred concentration on large numbers of cells

based on turbidity of diluted sample– Easy to use

• Disadvantages– Tendency to overestimate because other objects

(debris, somatic cells, etc) will contribute to increased turbidity

– Cannot evaluate concentration of semen extended in non optically clear media



Densimeter - ARSSpermacue - Minitube

BD Spec 20IMV - AccuRead

PHOTOMETRIC SPERM COUNTERS 2 NEW DIRECT COUNTING METHODS USED IN OUR LAB

• NucleoCounter SP100– Validated by hemacytometer at SBS– Validated by hemacytometer and flow

cytometry in several other laboratories• CASA

– Killed sperm with fixed depth chambers (Leja slides)

– Validated by hemacytometer and SP100 at SBS

AccuracyAccuracyLowLow HighHigh

PrecisionPrecision

HighHigh

LowLow

NucleoCounter SP-100

Chemometec NucleoCounter SP-100

How does it work? How does it work?

• Similar to flow cytometer• Counts fluorescently stained DNA particles

• Similar to hemocytometer• Counts cells in a fixed window/volume

• Propidium Iodide • Not permeable to intact membranes• Only counts dead cells

How does it work? How does it work?




v Sperm membranes are permeabilized by dilution in SP-100 reagent.

v Diluted sample is loaded into cassette and transported through the microfluidic channels.

v Cells are labeled with propidium iodide (PI) within the fluidic network. PI intercalates with cellular DNA and nuclei emit red fluorescence.


v Camera captures each cell nucleus as white dots on a dark background which are then counted by image analysis software.

Dilution factor in SP-100 reagent is important to maintain suitable cell concentration in viewing window.

NucleoCounter SP-100 NucleoCounter SP-100

v Reliable, fast and objective method for cell counting based on fluorescent microscopy and automated image analysis.

v Hansen et al (2006) with boar semen:

- Repeatability (CV) FACScount 2.7%

Hemacytometer 7.1%

Photometer 10.4%

Sperm Vision (CASA) 8.1%

Nucleocounter 3.1%

-Highest correlation between FACS count and Nucleocounter SP-100

y = 0.8436x + 4.0605R2 = 0.9567

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100

Nucleocounter

Hem

ocyt

omet

er

.

Correlation of Nucleocounter and Hemocytometer Correlation of Nucleocounter and Hemocytometer estimates of sperm concentration in extended and neat estimates of sperm concentration in extended and neat

bovine semen samplesbovine semen samples

y = 1.019x - 0.0647R2 = 0.9775

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5 3

Nucleocounter

Hem

ocyt

omet

er

.

Neat Semen (x 109)Extended Semen (x 106)


v Serial Dilution 25 - 100 x 106/mL (n=3)

Evaluation of Serial Dilutions on the Nucleocounter

R2 = 0.975

0

20

40

60

80

100

0 20 40 60 80 100 120Concentration

Cou

nt



NucleoCounter SP-100Stallion Semen

Nucleocounter vs Hemacytometer (n = 67 counts)

100

150

200

250

300

350

400

450

100 150 200 250 300 350 400 450Hemacytometer

Nuc

leoc

ount

er

Mean (n=67) Nuclecounter = 274 x 106/mL

Hemacytometer = 269 x 106/mL

R2 = 0.89

The “NucleoCounter”

• Accurate YES • Precise EXTREMELY, CV ~3.0%• User-friendly YES• Fast YES, ~30 s/sample • Economical ????

• >$14,000 Nucleocounter• >$3.00/Cassette

Motility

Common Milk Based Extenders for Stallion Semen

Defined Milk Protein-native phosphocaseinateHanks balanced saltsGlucose, Lactose

Skimmilk - glucoseSkimmilk-glucose-sucrose

EquiPro - Minitube INRA 96 - IMV

Defined milk protein-native caseinateWhey proteinsSugarsglycin

Visual Subjective

Computer Assisted Semen Analysis(CASA)

Motility Evaluation Temperature is CRITICAL!Water bath better than incubator for incubating motility samples



Motility Evaluation

• Dilute sample to 25-30 x106/ml permits visualization of individual sperm motion

• Incubate for 5 minutes at 37C

Objective Analysis of Semen Quality

Hamilton-Thorne CEROS (CASA System) Digital image of motile sperm (negative phase contrast)

Record of sperm tracks



IndividualSperm Track

Factors That Affect Results of CASA

• Instrument Settings– gates– define prog motility– slow cells motile?– framing rate and duration of analysis

• Sperm Concentration• Debris and Extender Clarity• Chamber Depth and Type

QUESTIONS ?

Infectious Disease Control in Cooled and Frozen Semen



“Of the various modes of transmission involved in the spread of infectious disease the venereal route is one of the most effective in ensuring the dissemination of specific pathogens.”

Dr. Peter Timoney speaking at American Association of Equine Practitioners Annual Meeting Baltimore , MD 2000

• Most pathogens that can be found in semen will withstand conventional cooling and cryopreservation protocols

Bacteria

• Psuedomonas aeruginosa• Klebsiella pneumoniae

– Capsule types 1,2,5,7?• Beta Streptococcus zooepidemicus• Taylorella equigenitalis

– Causative agent of CEM

Viruses• Equine arteritis virus (EAV)• Equine herpesvirus-3 (EHV-3)

– Coital Exanthema• Equine herpesvirus-1 (EHV-1)

– Recent report of EHV-1 detected in semen of 13% of 390 stallions screened with PCR with no clinical signs of respiratory disease

• Hebia-Fellah et al, Therio 71 (2009) 1381-1389

• Equine Infectious Anemia• West Nile Virus

Others

• Piroplasmosis– Protozoan parasites babesia equi & babesia

caballi• Mycoplasmas• Dourine

– Trypanosoma equiperdum – tissue parasite

Recent Outbreaks in North America

• EVA 2006– Confirmed in 10 states primarily in West– Primarily in QH population previously naïve to

EAV• 1998 study by NAHMS showed susceptibility• Only 0.6% of sampling of QH seropositive

– Index stallion – QH in new Mexico• Transported cooled semen around country• 70% of direct exposures resulted from use of this

stallion’s semen• Movement of ET recip and donor mares from index

farm to other states account for remaining 30% of exposures



EVA 2006

• Virus related abortion rates of 50-55% on some farms

• Several exposed stallions became carriers• No disease related fatalities in foals or

others• EAV circulated for 5-6 months (May –

November)

Recent Outbreaks in North America

• CEM 2008 &2009• Index stallion not yet identified?• First positive stallion discovered during CEM

testing prior to frozen semen export• Stallion was at a station with 20 other

stallions• Numerous stallions at this facility

subsequently found positive• No natural service, transmission appears to

be from collection equipment or phantom

Contagious Equine MetritisAs of June 2, 2009

• 21 positive stallions located in 7 states• 5 positive mares• 939 exposed horses located in 48 states

– 272 exposed or positive stallions in 29 states– 667 exposed or positive mares in 46 states– ALL currently under quarantine or on hold

order

Contagious Equine Metritis2008/2009 Outbreak

• Breeds of Positive Stallions– Appaloosa– Arabian– Dutch Warmblood– Fjord– Friesian– Hackney– Paint– Quarter Horse– Saddlebred– Thoroughbred

Economic Impact of CEM Outbreak

• 272 stallions quarantined and tested– Testing and clearing negative exposed stallion

• $5,000 x 250 = $1.25 million– Testing, treating, retesting & clearing positive

stallion $15,000 x 22 = $330,000– Loss of use of breeding stallion for nearly entire

2009 breeding season $$$$??• 667 mares quarantined and tested

– Testing and clearing negative mare• $1,000 x 667 = $667,000



Economic Impact of CEM Outbreak

Total direct costs for testing and treating exposed stallions and mares

$2.247 MILLION

So far……….. plusCost for loss of useCost of reputation for stallion

Recommendations

• Adopt a mandatory health testing protocol and codes of practice for all stallions being used in DOMESTIC transported cooled and frozen semen breeding programs based on Codes of Practice and requirements similar to EEC Directives for trade of semen between member States.

Recommendations

• Screen all breeding stallions at the beginning of each season for CEMO, Psuedomonas aeruginosa, klebsiella pnuemonia, beta strep

• Vaccinate all EAV seronegative breeding stallions and booster annually

• Perform virus isolation on semen of EAV seropositive stallions to determine carrier state

Recommendations

• Strict hygiene and biosecurity protocols for semen collection centers

• Advocate creating stocks of “disease-free”frozen semen by having stallions collected and frozen in approved stations with testing before and after freezing session– Allows for continuation of breeding season

even in the face of an infectious disease outbreak such as recent equine influenza in Australia

Research Summary

Cooled Bacteriology Trail with Dr. Althouse Results

v Contamination found in 31/ 47 (66% prevalence)

- 68% single contaminant

- 32% multiple contaminants

v Majority of contaminants were Corynebacterium spp and Bacillus spp (64%)

Frozen Semen

Industry Concerns and Considerations



Contracted Breeding or Single Dose Commodity Sale?

• What system is in the best interest of the industry?

• What are the considerations for stallion owners?

• What are the considerations for mare owners?

• What role do the breed registries play?

Mare and Stallion Owner Agreement

• Breeding Fee has two components– Cost of genetic potential from stallion

delivered through sperm• STUD FEE

– Cost of overhead associated with delivering the genetic material

• BOOKING FEE• LAB FEE• COLLECTION FEE• CHUTE FEE• HANDLING FEE• DELIVERY COSTS

GENETICS IS THE PRODUCT

STUD FEE

• ONLY HAS VALUE IF FOAL IS PRODUCED– Successful outcome of breeding is dependent

upon several factors• Inherent stallion fertility• Stallion management associated with collection,

processing and delivery of viable semen• Inherent mare fertility• Mare management associated with insemination

STUD FEE

• RISK ASSOCIATED WITH SUCCESSFUL OUTCOME SHOULD BE SHARED BY BOTH PARTIES

• STRAWS OF FROZEN SEMEN OR DOSES OF COOLED SEMEN HAVE NO INTRINSIC VALUE

OVERHEAD COSTS

• Several ways to deliver genetic material to mare– Live cover– Fresh Semen AI– Cooled Transported Semen AI– Frozen Semen AI

• Each of these systems has associated overhead costs• Each of these systems has associated advantages and

disadvantages to stallion owner and mare owner



What is a fair system for both parties?

• Mare owner pays costs associated with overhead to provide genetics for that mating– Cover with non-refundable booking fee paid in

advance• Stud fee for genetics paid only if foal is

produced

Frozen Semen as a Commodity

• Pros and Cons for Stallion Owners• Pros and Cons for Mare Owners• Why a Stallion is Not a Bull• Why This is Bad for the Industry• Is This What the Industry Wants Anyway?• What is Required to Make it Work

Stallion Owners

• Pros– Paid in advance– Not dependent on

mare management for result

– No need to track doses of semen

– No guarantee required– Mare owner assumes

all risk

• Cons– No control over semen

use– Potential for multiple

foals from one fee– Potential for lower

overall revenue– Damage to reputation

from misuse of semen• Splitting doses, etc

Mare Owners

• Pros– Potentially lower cost

to produce foal– Potential to produce

multiple foals for one fee

– Resale of unused doses

• Cons– No guarantee of

quality or pregnancy– No recourse if poor

quality– Potentially higher cost

to produce foal– Mare owner assumes

all risk

Why a Stallion is Not a Bull• Bull

– Highly selected for fertility (with frozen semen)

– Young bulls fertility tested before semen is sold

– Industry standards for commercial semen quality

– Low variability in semen quality and fertility

– Breeders assured reasonable chance of pregnancy

• Stallion– Little or no selection for

fertility– No fertility testing of

semen before marketing– Lack of industry

standards for commercial semen quality

– High variability in semen quality and fertility

– Breeders cannot assume reasonable chance of success

Why is this bad for the industry?• Mare owners trying to save money will purchase too few

doses, requiring increased vet costs and reducing chance of success

• Stallion owners worried that mare owners are splitting doses and using ART will reduce number of sperm per dose resulting in reduced pregnancy rates

• Due to frustration of mare owners and stallion owners, viable and efficient technology will be underutilized

• Domestic stallion owners unable to compete with inexpensive commoditized frozen semen prices



Is This What The Industry Wants Anyway?

What would it take to make this system work?

• Establish comprehensive standards for commercial quality frozen semen

• Establish 3rd party objective quality control and testing program for commercial frozen semen

• regulate foal registration to prevent abuse of ART by semen purchasers– AQHA, example

AQHA System• Foal Registration requires signed

Breeder’s Certificate (issued by stallion owner) and must correspond with mares listed on the Stallion Breeding Report which has been filed with the registry by the stallion owner/agent– This allows stallion owner to control the

number of registerable foals produced (and stud fees collected) from any shipment of frozen semen

AQHA System

• Retained Semen Rights Permit– In the event that a stallion owner wishes to

sell a stallion and retain the rights to use stored frozen semen to produce registered foals, he can purchase from AQHA Retained Semen Rights Permits

Retained Semen Rights Permit

• Can only be purchased by the current owner of the stallion

• Only one foal may be registered per permit• Once the stallion is sold, the former owner

can not purchase additional permits• The permit takes the place of the

Breeder’s Certificate for purpose of foal registration

Retained Semen Rights Permit

• The number of outstanding permits for any stallion will be tracked by AQHA and be a matter of public record

• Ownership of permits may be transferred



THANK YOUQUESTIONS OR COMMENTS?

Separation of X and Y Bearing Spermatozoa

Principle

• In the stallion, DNA Content of X bearing sperm is 3.7% higher than Y bearing sperm

• Sperm stained with flourochrome H33342• Further stained with food dye that

quenches fluorescence of non-viable sperm so only viable sperm are sorted

Sexing Technologies – Navasota, Texas

Sort Process• The sample is placed on the

sample station• The sample is then

pressurized to allow for a flow through the sample line

• The sample line joins with the sheath fluid line

• The sheath fluid is a compatible media that “envelopes” the sample stream

• The sheath fluid thus surrounds the sample (or core) stream and helps to orient the flow through the flow cytometer

Sort Process• The combine streams enter the

Cytonozzle™ which contains a piezo crystal• It has a very thin wall between it and the

fluid inside the nozzle that creates the stream

• An electronic wave is applied at frequencies close to 75khz

• The vibration created causes the combined stream to break off into well controlled droplets

• The diameter of the Cytonozzle™ is 70u which, with pressures reaching 40psi, allows for 70,000 drops to materialize every second

• With specific adjustments to the flow cytometer for determining the position of the “last attached drop,” the sperm sorter is capable of metering one sperm cell per drop formed

• The Cytonozzle™ then aids to orient the single cell within the drop as it exits the tip

• The drops enters the “air” and is excited by a focused UV laser beam

• The resulting fluorescence is captured through optical lenses and filters before reaching the detectors or PMTs



Sort Process

• The sperm sorter has two detectors – one measures forward (0°) fluorescence and the other measures side (90°) fluorescence

• The forward detector measures DNA content while the side detector measures cell orientation

• Optimum orientation occurs when the sperm cell aligns such that the flattened aspect of the head is fully facing the forward detector and laser beam while the narrow edge orients with the side detector

• This orientation creates the brightest fluorescence possible

Sort Process• Once information is received as to

the “sex” of the interrogated sperm cell within a given drop, the drop is then subsequently charged and as it drops between the high voltage plates it is deflected into a receptacle containing “catch fluid”

• “Catch fluid” is usually some type of extender with added nutrients to sustain sperm cell viability

• “Sexed” sperm cells that are positively charged are deflected toward the negatively charged high voltage plate and visa versa

• Both sexes can be sorted simultaneously

• Based on current technology, ~60-70% of cells interrogated can be successfully sorted with a high degree of accuracy

AI of Sexed Semen

• Sort rate very slow: would take ~20 hours to sort 300 million sperm with single nozzle

• Low dose (5 to 20 million sperm) deep uterine horn insemination required.

Fertility

• 72% conc rate for mares inseminated with hysteroscopic technique. 20 million sperm sorted after 15 hours storage for 18 hours

• Practical application requires use of sorted then frozen semen

Frozen Sorted Semen

• Limited results with sorted frozen semen• Lindsey et al

– 5 million sperm sorted PR= 13%– 5 million non sorted PR= 38%

• Clulow et al– 6,13 or 25 million sperm by hysteroscope – 9% PR for sorted-frozen– 20% for non-sorted frozen

Thank You for Your AttentionAny Questions?



Cost of Semen

Cost of Veterinary

Care

Unanswered questions

• Low-dose deep uterine horn insemination of frozen-thawed semen

• Rectally guided or videoendoscopic?• Why are we trying to inseminate mares

with far fewer frozen-thawed (potentially cryodamaged) motile sperm than is considered acceptable for cooled semen?

Thanks for your attention!

Research Summary

Cooled Bacteriology Trail with Dr. Althouse Methods

v 47 shipped in cooled semen samples collected by SBS affiliate labs

v 10µL cultured on 5% blood, brain heart infusion and MacConkey plates

v Colonies counted visually and multiplied by 100 to give CFU/mL

v Isolates identified by gram stain, morphology, biochemical reactions & PCR



Documents

ABDULLAH Cooled and Frozen Semen Technology Loomis - semen.pdf · Cooled and Frozen Semen Technology Quality Control and the Impact on the Breeding Industry Paul Loomis ABDULLAH 1984