1. WELCOME

2. EMBRYO TRANSFER IN BOVINE

3. Contents Role of protein supplementation: Has a bull a role in IVF?
Sperm concentration:
Effect of cumulus cells on IVF:
Role of glucose on IVF:
Effect of bovine serum albumen on Role of glucose on IVF:
Sperm-oocyte interaction:
How to assess efficiency of IVF:
Role of serum in in-vitro culturing Role of energy on IVC:
Developmental block:
Effect of embryo density on IVC:
Embryo evaluation:
Buffalo oocytes' IVM, IVF & IVC:
Costs.
Future ideas
Serum-free and Chemically Defined Media:
Hormone supplementation:
Role of somatic cells in IVM
Duration of IVM:
Signs of in-vitro maturation:
In-vitro oocyte fertilization (In-vitro sperm capacitation:
Use of fresh / frozen semen
Synopsis of bovine embryo transfer procedures.
In-vitro fertilization in bovine
Collection of Oocytes
Oocyte selection for IVM:
In-vitro Maturation Media:
The History.
What is Embryo Transfer?
Embryo Transfer as a Management Tool
Embryo Transfer offers farmers the chance
The Donor Cow.
The Recipient Cow.
Hormones.
Preparation.
The Flush.
The difference between surgical and non - surgical trans...
Conception Rate.
Temperatures that embryos can be stored in. :

4. The History.
The first successful Embryo Transfer took place in England in the 1890's by a fellow named Walter Heap, his subjects were rabbits.
Although that was a success, Embryo Transfer has not been applied commercially until the arrival of the hormone FSH, which stands for Follicle Stimulating Hormone, which occurred in the 1950's.
At first the only technique was surgical to both flush and implant the embryos.
While these methods were successful they were very expensive, required a large setup, and a lot of experience.

5. What is Embryo Transfer?
Embryo Transfer is basically, multiple injections of hormone to stimulate and multiply the ovulations in the cow that you want to get the embryos from
The donor cow is inseminated at normal time but 12 hours apart and 3-4 times
Seven (7) days later the rinsing out of the uterus to extract the embryos and ova (unfertilised, fertilised or degenerate)
Isolation of the good embryos using a microscope and then transfer into the recipient cows or frozen

6. Embryo Transfer as a Management Tool
Embryo Transfer is now accepted as the quickest and most cost efficient method of increasing the rate of genetic improvement within a herd.
For beef and dairy operations, it can be a valuable addition to any breeding program, leading to greater efficiency and profitability.

7. Embryo Transfer offers farmers the chance to
Produce up to ten or more progeny per year from their best cows.
Profit from the increased sale of quality genetics without losing the bloodlines.
Extend the productive life of some older cows, incapable of carrying another calf by producing further progeny through the use of embryo transfer.
Conserve the genetics in their herd through the uses of, embryo freezing for, export, domestic sale or future transfers on their own farm.
Introduce top genetics into the farmers herd, rapidly and economically from Australian and overseas.
The top cattle in any herd are rarely available for purchase, however the owners are often willing to sell embryos. The importation of new blood stock can be achieved safely and economically through the purchase of embryos from anywhere in the world, once the quarantine regulations have been fulfilled

8. The Donor Cow.
Any cow that is mature and cycling is able to be put through an embryo transfer program.
The only problem that could stop the cow or heifer from being flushed is abnormalities of the reproductive tract or with secondary abnormalities due to disease or injury.
Some heifers as young as eight (8) months have been successful in flushing embryos as have cows eighteen (18) years old. Although it is possible for embryos to be flushed from a cow or heifer at these ages the result obtained from the heifer or cow it may not be worth the effort.
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9. A major problem that has to be faced is that the response of a donor cow can vary from or more eggs, but the average being around depending on the breed, age and maturity.
Nutrition is a very important factor, and donors should be on a rising plain of nutrition in the month leading up to the flush. If the donor isn't in good condition then it will not produce as many eggs to be fertilized.
Also there is the response to the drugs, sometimes the cow will over respond to the drugs and sometimes under respond. Future programs would have the drug rates altered to suit the response.

11. Hormones. There are three hormones:
FSH, Follicle Stimulating Hormone, taken from a cow's Pituitaries
PG, Prostaglandin, and
PMSG, Pregnant Mare Serum Gonadotropin

13. Preparation.
First choose a donor cow for flushing, then insert a CIDR to stop the cow from cycling.
Give the cow a series of injections morning and night with 0.8 ml. FSH.
On the third day of injections also inject some Prostaglandin hormone causing the cow to cycle (come into season).
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14. After all injections have been done and the CIDR has been pulled, watch the cow to find out the time when it comes on heat.
Ten (10) hours after the cow has been on heat inseminate the donor cow with two straws of semen, then hours later a second insemination using one straw of semen, then another 12 hours later a third insemination and finally 12 hours after that another insemination if you wish.
Seven days later the donor cow is flushed and the embryos are isolated and then inserted into the cows or frozen.

16. The Flush.
The cow is placed in a head bail then the cow is cleaned out.
An introducer is put into the cow's vagina guided rectally and is pushed gently through the cervix into the uterus.
A tube called a catheter is put in through the shell of the introducer and pushed right up into the uterine horn.
A small balloon called a cuff at the end of the catheter is blown up.
After the cuff has been blown up, a fluid for lubrication and nutrition of the embryos has to be injected.
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17. The fluid is a sodium chloride based phosphate that has a small amount of serum.
About mls of this fluid is injected through the tube and into the cows uterine horn.
The fluid is then sent back through the center tubing of the catheter in to an embryo filter. The extra fluid is released while the embryos are kept in the filter by a small grid on the bottom
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18. After one side of the uterus has been flushed then the other side is flushed.
When the flush is complete the embryo filter is washed out into a small container.
The vets then search for embryos looking through a microscope.
The embryos are graded and put into straws and implanted into the recipient cow or frozen for later use.

31. The Recipient Cow.
A recipient is best to be a bit larger than the embryo calves mother so that it will not have any trouble having the calf.
Not all cows are able to be a recipient cow, before the cow or heifer can become a recipient it must be palpated to see if everything is in working order.
Some cows have a twisted cervix or have had calving problems so it is best to avoid using these cows or heifers for recipients.
Just before the flush the vet has to check the recipient cow or heifer to find out which side the cow has ovulated on so the embryo can be inserted on that side.
The recipient cow should be in good condition to have a good conception rate.

34. The difference between surgical and non - surgical transfers.
Surgical transfer requires a small cut in the cows flank on the side of the ovulation. The uterus is pulled out and a small hole is made to the uterus. A small catheter is placed in through the hole and the embryo released.
Non surgical is the same as A.I. A special gun is place up through the cow's vagina and pushed through the cervix. The embryo is placed in the side of the ovulation and released from the gun.

35. Conception Rate.
With embryo transfer an already fertilized egg is being inserted, so the conception rate should be % higher than artificial insemination, but a lot depends on the condition of the recipients.
On an A.I. program the conception rate is % while on an embryo transfer program it is expected roughly the same sometimes a bit higher

36. Temperatures that embryos can be stored in.
Fresh embryos can be stored at 37ºC for hours without much harm.
It is recommended that the embryos are inserted into the recipient cow or frozen as soon as possible.
The embryos are frozen in liquid nitrogen, they can be stored in this for ever.

37. Costs. The costs of an embryo transfer program is fairly expensive
The hormones and drugs are approximately $200 per donor.
On a per donor basis you are looking at spending approx. $100 - $300 for the flush procedure.
Transfer fees per embryo vary between $95 for non surgical to $150 for surgical.
Cost of providing good quality recipients should not be over looked. This averages out to around $300 - $500 per recipient.

38. Future ideas.
With the advancement in embryo transfer, scientists are taking embryos and breaking them up into individual cells and making identical animals, this method is called cloning.
The cloning aspect is not commercially available yet but should be soon. It really depends on the imagination of the people that are involved with the research.
This may lead to farmers taking embryos out of the tank, seven days after examining their own recipients and then transferring the embryo.

39. Synopsis of bovine embryo transfer procedures.

40. IN-VITRO FERTILIZATION
IN BOVINE

41. History
Proper in-vitro embryo production is important not only forinexpensive mass production of embryos for transfer in domestic animals, but is likely the foundation in which more advanced technology (cloning, sexing, genetic engineering) are dependent.
Since bovine IVF became possible in the early (1980s), a lot of efforts have been done to clarify the crucial steps in IVM & IVF.

42. Collection of OOcytes:
Dissection of ovarian follicles.
Aspiration of ovarian follicles.
Slicing the ovaries.
Puncturing visible surface follicles.
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43. In cattle, aspiration of vesicular follicles is most commonly employed for oocyte retrieval.
In buffalo, slicing is a simple effective method for collecting a high buffalo oocyte yield of in-vitro culturing
Ovaries containing CLs have yielded lower numbers of oocytes than those without CLs.
The feasibility of oocytes retrieval by vaginal ultrasound, guided follicular aspiration for IVM & IVF, has been established.

44. Oocyte selection for IVM:
On the basis of visual assessment of morphological features by the presence of:
An intact complement of cumulus oophorus cells (COCs) surrounding the oocyte.
A homogenous appearing ooplasm.
Oocytes obtained from 6-7 mm follicles, having many layers of cumulus cells produced significantly higher proportion of morula / blastocyte stage embryos (65.9%) compared to those obtained from 2-6 mm follicles (34.3%).

45. In-vitro Maturation Media:
To enhance the complete and functional maturation of cow COCs in-vitro, various factors have been investigated:
Maturation media.
Protein & hormone supplementations.
There are no proven media that allow oocytes to develop without harm.
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46. Estradiol & gonadotrophins (FSH & LH).
One of most widely employed complex media for IVM of bovine oocytes is:
Tissue culture media-199 (TCM-199).
Ham's F-10.
In-vitro maturation of bovine oocyte is enhanced by supplementing the maturation media with:
Estrous cow serum.
Follicular fluid.
Fetal bovine serum.
Estradiol & gonadotrophins (FSH & LH).

47. Role of protein supplementation:
To prevent hardening of zona pellucida and therefore preserving ability of matured oocytes for fertilization.
TCM-199 plus 20% BES supplemented with FSH, LH & E2 and without supplementation showed a maturation rate of 81.7% and 47.4%, respectively

48. Serum-free and Chemically Defined Media:
To avoid stickness of embryo that occurs when protein –free medium is employed, serum or bovine serum albumen have been replaced by synthetic macromolecules such as:
Polyvinyl-alcohol (PVA) or
Polyvinyl-pyrolidone (PVP).

49. Hormone supplementation:
Oocytes matured in-vitro in the presence of gonadotrophins and estradiol showed high maturation and fertilization rates compared to those matured without hormones.
LH markedly stimulates glycolysis and tricarboxlyic acid (TCA) cycle, while FSH increases glucose oxidation via pentose cycle.
The primary gonadotrophin responsible for in-vitro oocyte maturation and cumulus cell expansion is FSH.
Estradiol supports cytoplasmic maturational changes necessary for fertilization and early post-fertilization development.
High estradiol concentration has a negative effect on spindle formation and polar body extrusion.

50. Role of somatic cells in IVM:
The presence of cumulus cells is necessary for cytoplasmic and/or nuclear maturation of bovine oocytes.
The cumulus oophnorus acts as a bridge between the granulose membrane and the oocyte allowing the transfer of metabolites and signal molecules from the larger granulose cells to the oocyte.
Adding granulose cells to the maturation media improved the ability of bovine oocytes to form a male pronucleus and to develop into the blastocyst stage.
The good oocyte with compact and dense cumulus cells cleave significantly higher than those with thin cumulus layer or poor marked oocyte.

51. Duration of IVM:
During maturation, there is a germ vesicle break down (GVBD) expressed by extrusion of the polar body.
Extr4usion of the polar body occurs in 80% of oocytes between 12 and 18h of culturing.
Oocytes that extruded polar body h of culturing showed significant higher rates of cleavage and development.
IVM culture period to GVBD was related to the thickness and compactness of COCs with an apparently 6h delay in heavily compacted COCs.
24 h maturation period is superior for cattle and buffalo oocytes' maturation in-vitro.

52. Signs of in-vitro maturation:
The cumulus cells expand to form a spherical mass in three dimensions and the COCs appear to float in the culture dish.
There are changes in the dimensions of perivitelline space, expulsion of the first polar body and the establishment of the second metaphase spindle to the vitelline surface.
The ability to decondense the fertilizing sperm head has been used as an indication to the cytoplasmic maturation.
The only valid guide to complete oocyte maturation is the ability of the oocyte to cleave after fertilization.

53. In-vitro oocyte fertilization (IVF):
Successful in-vitro fertilization of oocytes requires:
maturated oocytes
optimal number of fertilized spermatozoa with vigorous motility
favorable culture conditions

54. In-vitro sperm capacitation:
It is a sequence of biochemical and physiological changes in the outer sperm membrane that enable the sperm to undergoes the acrosomal reaction in response to the zona pellucida.
Acrosomal reaction involves:
Fusion and breakdown of the sperm plasma and outer acrosome releasing the acrosomal enzymes.
Hyper activation of sperm motility that normally occurs near and/or on the zona pellucida.
A change in the sperm membrane permeability to calcium appears to be the primary signal for acrosomal reaction.
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55. In-vitro capacitation of bull spermatozoa has been achieved by incubation of fresh semen in:
Heparin (10 – 20 ug/ml).
Bovine follicular fluid
Calcium ionophore A-23187
Caffeine-benzoate (5 – 10 mM).

56. Use of fresh / frozen semen:
Fresh semen requires a longer in-vitro capacitation period than frozen-thawed semen.
Fresh semen gives better penetration rate than frozen–thawed semen.
Frozen-thawed semen is likely to deteriorate more rapidly than fresh semen.

57. Has a bull a role in IVF?
There is a great variation in penetration rates (from 35 to 96%) between different bulls.
Bull difference results in not only the fertilization process but also the subsequent embryonic development.
The importance of minimizing or eliminating the bull variance in IVF has been recommended.

58. Sperm concentration:
Adjustment of the sperm concentration is essential to avoid polyspermic fertilization.
In cattle IVF, the commonly used sperm concentration ranges from 0.5 to 5 x 106sperm / insemination dose.
Addition of sperm density of x106 sperm/ml in volume of 2-4 ul to 45 ul fertilization droplets suggests to yield optimum fertilization.

59. Effect of cumulus cells on IVF:
Directly enhance sperm function
Avoid polyspermy.
Facilitate oocyte transport.
Guide spermatozoa toward the oocyte.
Induce acrosome reaction in bovine spermatozoa.
Fertilization rate of oocytes with cumulus cells is significantly higher than that of denuded oocytes

60. Role of glucose on IVF:
Bovine spermatozoa could capacitate and penetrate oocytes at higher rates in the presence of glucose when caffeine and heparin were added to the IVF medium.
The effect of glucose can be reversed by phosphodiestrase inhibitor such as caffeine.
In the presence of glucose, fructose or other seminal sugars, the acrosome reaction of spermatozoa may be retarded until the environment is riched in lactate and pyruvate.

61. Effect of bovine serum albumen on IVF:
Exposure of bull spermatozoa to bovine serum albumen (10 mg/ml) is an appropriate physiological salt solution can result in capacitation in-vitro.
Serum albumen may play a key role in removing cholesterol and / or zinc from sperm during capacitation

62. Effect of penicillamine, hypotaurine & epinephrine (PHE):
Addition of PHE to the fertilization medium yielded a 23% increase in the cleavage rate by increasing the sperm motility, the percentage of spermatozoa that undergo acrosomal reaction and penetration rate of oocytes

63. Sperm-oocyte interaction:
Activation of the oocyte by the presence of spermatozoa.
The vitellus shrinks in volume, expelling fluid into the perivetilline space.
The sperm head in the vetillus swells and acquires the consistency of gel forming a male pronucleus.
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64. The sperm head decondensation occurs within 1 – 24 h of oocyte penetration and the pronucleus develops after a further 3 – 5 h.
There was a significant increase in rates of penetration and fertilization from 66% to 93% after 8 – 24 h sperm – oocyte incubation.
Oocytes and associated cells produce and secrete substances promoting fertilization

65. How to assess efficiency of IVF:
The evidences of successful in-vitro fertilization are:
Penetration of the sperm into the ooplasm.
Swelling of the sperm head.
Pronucleus formation.
Morphologically normal cleavage.
Blastocyst formation.
Breakdown of cortical granules.
Evidence of a sperm tail in the ooplasm

66. Role of serum in in-vitro culturing (IVC):
Serum has a biphasic effect, suppressing early and stimulating later embryo development.
Addition of serum to culture media significantly promotes blastocyst formation of bovine embryo.
Serum may contain an embryo trophic factor (s0 that promote proliferation and cell differentiation in-vitro and/or removing the toxic effects of an unsuitable medium.

67. Role of energy on IVC:
Total glucose utilization in bovine embryo was low for the 6-16 cell stage and increased drastically at the morula stage.
Glucose at a higher level (5.6 mM) might have a latent harmful effect, even at the morula stage.
Glucose at a lower level might be beneficial for the embryonic development in-vitro.
Addition of lactate and pyruvate enhance the oocyte development to morula stage; a proper lactate / pyruvate ratio is essential for diminishing the oxidation-reduction potential of embryo

68. Developmental block:
When embryos are cultured on inappropriate conditions in-vitro, they encounter a developmental block at:
the 2 – cell stage in mice and hamster
the 4 – cell stage in pigs
the 8-16 cell stage in sheep and cattle
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69. The cell blocks can be overcome either by;
using specific compounds or
changing in medium composition by addition of:
bovine oviductal epithelial cells (BOECs).
Cumulus cells.
Granulose cells
Uterine cells
Trophoblastic cells

70. Effect of embryo density on IVC:
Several studies have shown improved development with increased embryo density.
It has been recommended that embryo density of one ovine embryo per 20 ul of culture media is superior for embryo development.

71. Embryo evaluation: Embryo morphology and survival correlate well with:
Measures of enzyme activity.
Glucose uptake.
Live-dead stains
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72. morphological criteria commonly used to evaluate embryo quality include:
Shape and color.
Compactness of cells.
Size of perivitelline space.
Number of extruded polar bodies.
Degenerated blastomeres.
based on the morphological features, embryos can be classified into four categories: excellent; good; fair; poor.

73. Buffalo oocytes' IVM, IVF & IVC:
In series of experiments on in-vitro maturation, fertilization and development of buffalo oocytes, it has been concluded that:
Oocytes recovered from ovaries with medium sized follicles enhanced more in-vitro embryo production than the small or large follicles.
The optimum time for oocyte recovery post slaughter of the animal ranged from 3 to 4 hours.
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74. Although the yielded number was little, the aspiration technique provided better quality oocytes than the mincing technique.
Oocytes surrounded with multi-layers of cumulus cells seemed to be of good quality and improved the in-vitro embryo production.
Using Ham's F-10 and TCM-199 proved to be effective in in-vitro buffalo embryo production.
The best maturation period to enhance in-vitro buffalo oocytes was hours.
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75. Supplementation of maturation medium with 10% FCS or 10% BES as well as FSH, LH & E2 provided more in-vitro maturation, fertilization and subsequent embryo production.
Best results were obtained with fresh buffalo spermatozoa capacitated in-vitro with heparin + caffeine at 2 – 3 xl06sperm/ml.
Culturing zygotes in groups (5-15/drop) in culture medium without glucose and FCS supplementation during the first 24 h of culturing, then with glucose (1.2mM0 and FCS (5%) supplementation after the first 24 h improved significantly the in-vitro buffalo embryo production.

76. THANK YOU
Prof.Dr. A.I.El-Azab Dept. of Theriogenology Fac.Vet.Med.,Zag.Univ./Benha