Artificial Reproductive Technologies I: insemination Cinzia Allegrucci
LO s List the main advantages and disadvantages of artificial insemination (AI) Explain methods for evaluating volume and concentration, motility and morphology of an ejaculate Consider the principles of species-relevant recommendations for AI Outline the main properties of semen diluents Give examples of diluents in routine use for short term storage of semen Outline the process by which sperm is frozen and stored
What is AI? Placing semen into the female reproductive tract Semen can be: fresh fresh-extended extended-chilled extended-frozen-thawed
Why is AI used? To allow greater access to superior genetics and maximise genetic improvement To reduce mating costs To reduce mating risks To control reproductive diseases To allow use of dead or injured sires As part of embryo transfer (ET) regimes
Advantages of fresh (extended) semen in AI May be convenient in breeding management / time May allow splitting of semen dose Reduces chance of female injury May reduce disease transmission (especially fresh-extended) Allows scrutiny of sperm quality and numbers prior to insemination
Advantages of transported semen in AI (chilled or frozen-thawed) Convenience of maintaining the female at home Avoids the stress and cost of shipping a female Reduces costs for female care at other facilities Decreases chance of injury to female during transport Reduces likelihood for transmission of diseases between premises Access to superior males which otherwise are not available to female owner Increases the genetic pool by permitting wider use of males Allows scrutiny of sperm quality and numbers prior to insemination
Disadvantages of transported semen in AI (chilled or frozenthawed) Fertility of preserved sperm is generally reduced (large species variations here) Insemination dose often must be increased c.f. fresh insemination Expenses and time commitment associated with shipping semen Increased intensity of female management is required to ensure transported semen is ordered and inseminated at an optimal time Increased technical demands for transported semen and the potential effect of poor technique on fertility requires heightened knowledge and skill of manager and veterinary surgeon
Semen examination This is only part of a breeding soundness examination May be undertaken When lowered fertility is suspected When abnormal sexual behaviour is seen Before sale Before the breeding season If a pathogenic infection is suspected (to enable culture/isolation of pathogens)
Semen collection Teasing Then collection using: Artificial vagina with teaser or dummy female (bull, stallion) Teaser or dummy female and manual manipulation of the penis (boar, dog) Electro-ejaculation (ram)
Semen evaluation Is there any gross abnormality? How many sperm are there? Do the sperm have normal motility? Do the sperm have normal morphology Can the sperm fertilise?
Is there any gross abnormality? What is the colour of the semen compared with normal? Is there normal fractionation? Is there a normal gel component?
How many sperm are there? Total sperm output calculated by multiplying semen volume and semen concentration Volume normally measured by collection into a graduated non-toxic container
How many sperm are there? Concentration normally measured by placing sperm into a haemocytometer counting chamber Dilution of sample with distilled water with detergent Kills sperm and allows counting Prevents clumping Or a densitometer without dilution
Do the sperm have normal motility? Remember sperm motility is temperature dependant Two methods of assessment Subjective assessment This is a standard method Objective assessment Computer aided
Do the sperm have normal motility? Place standard volume onto a warmed slide
Do the sperm have normal motility? Subjective Assessment Estimate the percentage of sperm with different motility types: 0 I II III IV Non motile Motile but not progressive Small circles with slow progression Large circles with moderate progression Vigorous forward progression
Do the sperm have normal motility? Fresh Dog Sperm 0 I II III IV 5 5 5 10 75 =95% =75% Frozen-Thawed Dog Sperm 10 5 10 50 25 =25% =90%
Do the sperm have normal motility? Objective Assessment Computer Assisted Sperm Evaluation (CASA) measures: Mean sperm velocity Mean sperm linearity Lateral head deviation Mean curvilinear velocity
Do the sperm have normal morphology? Requires highlighting of sperm (background staining) or staining of sperm Can also assess membrane function Combination of two stains most appropriate Nigrosin (background stain) Eosin (vital stain)
Do the sperm have normal morphology? With nigrosin:eosin Sperm with damaged membranes allow eosin to penetrate -> sperm appear pink Sperm with intact membranes exclude eosin -> sperm remain unstained (white)
Sperm abnormalities Abnormalities can occur on the head, midpiece and tail Thay can be primary (defect of spermatogenesis) secondary (epididymis) tertiary (post-ejaculation) Abnormalities include: pyriform head, knobbed acrosome, detached or swollen acrosome, nuclear vacuoles, detached heads, distal midpiece reflex, looped tail, coiled tail, cytoplasmic droplets
Knobbed acrosome Midpiece reflex Pyriform head Coiled tail Nuclear vacuoles Detached head Cytoplasmic droplets
Do the sperm have normal morphology? Summary 72% live normal sperm 84% total live sperm 16% dead sperm
Can the sperm fertilise? Conventional evaluation cannot give this information and relies upon the fact that if there are sufficient number of morphologically normal and motile sperm then fertility will be normal Fertilisation assays Zona binding test Oocyte penetration test
Semen characteristics of different species Semen characteristics of dogs: Second Fraction volume 0.5 to 2.0 ml Sperm concentration 150 to 600 x 106 /ml Total sperm output These are for example and need not be learned you are expected to know semen volume for different species Average 300 x 106 sperm Morphologically normal sperm Greater than 70%
Semen characteristics Characteristic Bull Ram Stallion Boar Dog Volume (ml) 4 1 60 (2-10) (0.5-2) (125500) 250 (125500) 10 (2-19) Fractionated N N Y Y Y Density (x106/ml) 1250 (6002800) 2000 (12503000) 120 100 (30-600) (251000) 300 (20540) Motile sperm % >70 >90 >60 >60 >85 Normal sperm % >85 >60 >60 >90 >75
Semen preservation Fresh-extended (room temperature) Diluted / cooled (5oC) Diluted / frozen (-196oC) then thawed
Life-span of preserved semen Depends on species but: Fresh-extended (room temperature) Up to 8 hours Diluted / cooled (5oC) Up to 4 days (many) to 10 days (boar) Diluted / frozen (-196oC) then thawed Indefinite
Dilution of semen Extender solutions aims to: protect sperm during cooling/freezing/warming supply an energy source to sperm maintain ph, osmolarity and ionic strength Prevent bacterial growth Factors important in semen preservation: Protective agents (milk or egg protein / glycerol) Sugars ph, osmolarity, ionic strength Antibacterial agents
Method of use, for example, fresh stallion semen Sample collection Filtration to remove gel Sample evaluation Dilution and splitting into several doses Insemination
Method of use, for example, chilled stallion semen Sample collection Filtration to remove gel Sample evaluation Dilution with extender solution: Non-fat dry milk (marvel) 2.40 g Glucose 4.90 g Sodium bicarbonate 0.15 g Deionised water up to 100 ml Penicillin 150 000 IU Streptomycin 150 000 mg You only need to recognise this is based on skimmed-milk
Method of use, for example, chilled stallion semen Shipping in temperature controlled device Sample evaluation Insemination
Freeze-thawing of semen Freeze-thawing can cause significant sperm damage and additional cryoprotectants are required: Penetrating (Glycerol, DMSO) Non-penetrating (proteins, large mwt. sugars) A controlled freezing rate is essential (and a specific thawing rate is important)
Influence of freezing rate If freezing rate is sufficiently slow and cell permeable to water During freezing Water leaves cell which progressively dehydrates Small ice crystals form inside cell On thawing Ice crystals melt and cell rehydrates and is functional If freezing rate is too rapid or the cell is impermeable During freezing Cell does not dehydrate Large ice crystals form inside the cell = cell is damaged On thawing Cell non-functional If freezing rate is too slow During freezing Cells dehydrate and solute concentration increases Solutes tend to precipitate (solution effect) Cells shrink beyond a minimum compatible with survival = cell is damaged On thawing Cell non-functional
Method of use, for example, frozen-thawed stallion semen Sample collection Filtration to remove gel Sample evaluation Dilution with extender solution Centrifugation to form sperm pellet concentrates sperm removes seminal plasma (Re) Suspend in freezing buffer to known sperm concentration
Method of use, for example, frozen-thawed stallion semen Slow cooling to 5 C Equilibration period (2-4 hours) Load in 0.5, 1.0 or 4.0 ml straws (vials / plastic bags also used) Freeze at standard freezing rate in liquid nitrogen vapour Plunge into liquid nitrogen
Method of use, for example, frozen-thawed stallion semen Storage in liquid nitrogen storage time (2000 to 4000 years to kill 63% of cells) Thaw rapidly For 0.5 ml straws, generally thaw at 37 C Plunge straws into water bath Carefully dry Snip ends and empty into nontoxic test tube
Species differences AI procedures vary in different species
Bulls Semen usually collected with artificial vagina Semen freezes well Uterine insemination easy after training Success similar to natural mating 50% calving rate to single insemination
Rams Laparoscopy Reasonable survival after freezing Trans-vaginal uterine insemination difficult so either cervical or laparoscopic uterine insemination used Success rates depend upon site of insemination 40% lambing rate for cervical insemination of nonfrozen semen 70% lambing rate for uterine insemination of non-frozen semen
Boars Poor freezing ability so most is extended in 50 ml bottles shipped at ambient temperature not cooled Uterine insemination easy Success similar to natural mating (80%) 75% farrowing rate to two inseminations at one oestrus
Stallions Poor freezing outcome and significant individual variation Fresh Chilled semen shipped in plastic bags in thermos Frozen semen stored in 1.0 or 4.0 ml straws Uterine insemination easy Success rates Natural mating (75% conception) Chilled semen AI (65% conception) Frozen-thawed semen AI (45% conception)
Dogs Poor freezing outcome and significant individual variation Uterine insemination difficult but possible Success rates Natural mating (90% conception), Chilled semen AI (65% conception), Frozen-thawed semen AI (50% conception)
LO s List the main advantages and disadvantages of artificial insemination (AI) Explain methods for evaluating volume and concentration, motility and morphology of an ejaculate Consider the principles of species-relevant recommendations for AI Outline the main properties of semen diluents Give examples of diluents in routine use for short term storage of semen Outline the process by which sperm is frozen and stored