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Semen Collection and Freezing
The collection and freezing of ram and buck semen is an Australian technology that was developed in the 1960’s, and is now a very routine tool used by stud breeders to preserve genetics. Semen, once successfully frozen, will last forever. LBS has been freezing semen since 1983, and operates to AQIS export standards. Jerilderie is an AQIS accredited export semen collection shed.
Why Freeze Semen?
Semen is frozen for one or a number of the following reasons:
- As insurance against the loss or infertility of a valuable sire.
- For the purpose of making semen available for sale to other breeders.
- To deliver “semen shares” in rams that have multiple owners.
- To send semen a long distance, interstate or international.
- To use a sire in an AI program that cannot be used for natural joining, such as Show rams, very young rams or infirm rams.
What sires can be collected?
Nearly all sires are candidates for semen collection, and even some injured or arthritic sires can be collected successfully. The health of the testicles is the major limitation to successful semen freezing, and that functionally remains largely unknown until semen is collected and analysed.
Semen from rams with significant apparent testicular pathology can be frozen successfully, whilst some rams with no apparent problem can be infertile. A single functional testicle can produce fertile semen.
Age is no particular problem, if testicles are functional and healthy. As young as 8 months and as old as 14 years have given viable semen in enough volume to be freezable.
How is it done?
There are two methods of semen collection; byartificial vagina (AV) or electro-ejaculation.
The usual method is collection by AV. This is because it is reasonably simple to train most sires, they can give multiple ejaculates on a single day, quality at collection is on average better than electro-ejaculated semen, and it is much less labour intensive for the AI centre than electro-ejaculation.
AV collection involves placing a teaser ewe or doe in a restraining bail, introducing the sire to the teaser, and when the sire mounts his penis is guided away from the teaser and into the AV. With the AV at the appropriate temperature and pressure the sire will ejaculate almost immediately, and a collection glass in the AV collects the ejaculate. With patience and quiet handling most sires collect within a few days, and some collect immediately. LBS has been training rams since 1983….. so it is routine.
The teaser does not have to be cycling for most sires to attempt to mount. It sometimes helps some shy rams or bucks if the teaser is in oestrus, which can be done artificially with a hormone injection.
Electro-ejaculation involves the solid restraint of the sire and a rectal probe, through which a steadily increasing current is passed until ejaculation occurs. Not every sire will give semen by this method. If the sire is not handled gently, or too much power used, he will usually urinate in the semen collection glass, which renders the semen unusable. Thus, electro-ejaculation is only performed when absolutely necessary and then with considerable care.
Electro-ejaculation does procure semen from sires that will not serve ewes with a handler close at hand, sires that have a physical injury preventing natural service and sires that have a penile injury that prevents use of the AV.
What does freezing of the semen involve?
Each ejaculate, after collection, is immediately assessed for both density and motility. If either of these factors is lower than our set standards, the semen is discarded at this point.
If the semen is assessed as freezable, then it will be extended with a chemical dilutent designed to protect the semen during chilling and freezing, and supply energy for the sperm during the periods of activity (pre-freeze and post-thaw). Using either dry-ice (frozen carbon dioxide) or liquid nitrogen vapour the semen is taken through freezing point, and then plunged quickly into liquid nitrogen (-196 degrees C).
Ram and buck semen is frozen and stored in either pellets or straws. The pellet is a small pellet consisting of just diluted semen, and is created on indentations made on dry-ice. Straws are the standard .25ml plastic straw, designed for cattle semen freezing and use. The pellets is a standard 3 ewe doses and the straw is a single dose.
Each ejaculate is assessed again after freezing. A pellet or straw is thawed, and microscopic assessment of the semen is done by a trained assessor. Without training, the very subjective decision of the percent alive is virtually meaningless. Assessors judge the percent of forward motile sperm, and looks for any spermatic abnormalities. Strict limits are applied to both factors, and the Australian sheep artificial breeding industry has worked hard to set meaningful standards that ensure the semen released to the industry is never a limiting factor in AI programmes. Semen that is below standard is discarded.
Frozen semen is kept permanently in liquid nitrogen tanks, and LBS has an inventory system that will show you all movements, each quarter. No semen is despatched or discarded without the owners instructions.
How often can sires be collected?
An average ram or buck may give .5ml to 1.5ml of semen per ejaculate. Some sires can do this 2 or 3 times each day, and perhaps every day of the week. This very much depends on the individual. The range is large and there are a number of reasons why the collections may be limited. These include:
- A lack libido to perform multiple serves.
- Volume of the ejaculate drops until the volume is unusable.
- The density of the semen starts to drop, also making it unusable.
- Only the very best quality semen will survive freezing and thawing.
Given that one semen ejaculate will dilute to givemaybe 60 to 80 doses of frozen semen, and that an average ram will give us say 2 ejaculates on 3 days per week, 400 to 500 doses per week is an average expectation. This ranges from nil to 1000 doses.
Will semen collection affect the reproductive health or capacity of the ram?
No. As sires are actually only working very lightly at a collection centre, and their fertility is being assessed constantly, most sires leave a centre in better reproductive health than when they arrive.
In the paddock situation, if a sire is joined to 50 females, he will have about 3 cycling each day for say 17 days. If he serves each female 3 times, after a few days his semen volume and density has dropped to the point where it would be quite unusable for freezing, but is adequate to get females pregnant. Sires rapidly lose body condition and testicle mass during normal paddock joining.
Can frozen semen transmit disease?
Two venereal diseases are tested for in sheep, and hence avoided, at LBS Collection sheds. These diseases are Brucella ovis (the cause of Brucellosis in sheep) and Actinobacillus seminis. If a ram has either of these diseases, his semen is potentially infective. Ewes inseminated with the infective semen can then pass these diseases onto any rams that come into contact with them, particularly the back-up rams used after the AI.
Thus it is important to use credible professional inseminators, and to ensure that semen purchased is from licensed artificial breeding centres.
LBS Collection Centres
LBS has 2 centres (Jerilderie and Yass) and will collect and freeze the best possible semen from your sire. Disease control measures and management with over 25 years experience in operating semen collection will ensure no risk to your sire. LBS owns no sires, nor any stud animals, and guarantees that you, the owner, retains all control over your sires genetics at all time.
Laparoscopic Insemination of Sheep and Goats
This technique of artificial insemination involves the synchronisation of oestrus and a single, timed insemination of ewes or does with a minor surgical procedure. The laparoscope is a rigid fibre-optic telescope, which is passed through the abdominal wall. This allows the operator to locate the internal reproductive tract and then inject the semen into the lumen of the uterine horn.
The technique bypasses the cervix of the ewe, which is narrow and tortuous and a barrier to simpler techniques of artificial insemination. The cervix of the doe is less of a barrier, but a more reliable result, with a smaller dose of semen can be achieved with the laparoscope than with cervical insemination.
Most laparoscopic AI utilises thawed frozen semen that has been stored in liquid nitrogen. Semen can also be collected fresh from a sire on the day of AI, extended with a diluent, and then inseminated. A normal ejaculate can inseminate up to100 ewes, and a sire may give 3 or 4 ejaculates on a day.
Why use Artificial Insemination in Sheep?
Using fresh semen, situations where laparoscopic insemination is used are:
- Breeding a much larger number of ewes to a valuable sire than is possible with natural service.
- Use of a very young sire, especially where his growth is not to be slowed by paddock joining.
- Use of an old or infirm sire, where fertility for the period of joining is doubtful.
- Use of show animals where body and fleece condition must be maintained.
- Use of an outside sire where disease control denies paddock use; semen can be collected off site and transported to the AI.
- Use of a sire where semen will not freeze satisfactorily, but is useable fresh.
Most laparoscopic insemination utilises frozen semen, for some of the following reasons:
- All of the above reasons applied to fresh semen (other than the last) are applicable.
- Purchase of frozen semen of a valuable sire.
- Use of a dead sire.
- Utilisation of a share in a sire bought in partnership.
- Use of a sire that is geographically distant; interstate or international.
- Used when fresh semen collected on the day of AI is infertile or insufficient.
Laparoscopic insemination using either fresh or frozen semen is also the standard method of fertilising embryos in routine MOET (Multiple Ovulation and Embryo Transfer) programmes.
For the insemination of a group of ewes or does to take place on the same day, the oestrus cycle of the group must be manipulated so that ovulation occurs at a set time.
Synchronisation is achieved by the use of a hormone containing intra-vaginal device and an injection of another hormone, PMSG (Pregnant Mare Serum Gonadotrophin). The intra-vaginal devices are in the form of a ‘sponge’ or a plastic ‘CIDR’ (Constant Internal Drug Release) which both contain progestagen. Whilst in the vagina, they slowly release the active hormone, stopping the normal oestrus cycle. When the device is removed after 12-14 days (longer in goats) and PMSG injected, the females all begin to cycle. Oestrus will occur 30-42 hours following removal of the sponge or CIDR with ovulation 50-62 hours post-removal. Ewes will cycle slightly earlier with CIDRs compared to sponges.
The injection of PMSG has two significant effects in females approaching follicle formation (the step in the cycle prior to ovulation). Firstly, it causes a narrowing of the time span over which animals in the group will all ovulate. Secondly, it will cause an increase in the number of ovulations each female has. This is seen as a higher percent of twins. With AI, there will be an increase in the twinning rate of approximately 20%. This increase can be controlled by adjusting the PMSG dose
With control over the ovulation of the females, insemination of the group can be done at a set time. This is done at 48 to 56 hours after device removal, with some variation between different devices and dose of PMSG.
The females are taken off feed and water the afternoon before the insemination to reduce rumen fill.
Just prior to the AI, the does/ewes are tranquillised with a sedative/analgesic and then loaded into the AI cradles.The lower belly area is prepared by removing the wool. The AI operator then uses two trochar and cannulae to penetrate the abdomen. Through one of these, the laparoscope is used to visualise the uterus in the pelvic cavity. Through the other, the insemination pipette, containing the semen is passed. The semen is placed inside the uterus by penetrating the wall of the uterus with the sharp tip of the insemination pipette.
Following topical treatment of the abdominal wounds, the female is promptly released from the cradle, and she can return to feed immediately.
A skilled operator will take approximately one minute to inseminate each animal. The professionalism and skill of the inseminator are vital components for success. The surgical approach, cleanliness, recognition of internal and uterine abnormalities, and risk assessment of infection and disease must be observed.
For at least 60 days after the AI, females should not have any management procedure performed. After this, the pregnancy is reasonably strong, but remember that any drench or drug used that is embryo toxic can be devastating.
Body condition is important. Animals should be presented at AI in condition score 2 - 3 for AI, and rise slowly from the time of insemination to score 3 - 4 over the next 30 days.
Diet is important. High protein feeds should be avoided at AI and for the following month. This recommendation is based on anecdotal evidence, and extrapolation of research on dairy heifers. We suggest that lucerne and clover dominated pastures should not be used, and cereals not legumes should be used as supplements following AI.
Careful management is needed through to the end of lambing. Avoid letting females become over-fat at any stage, but have good feed during the lambing period. Ewes lambing to a one day AI program will lamb over 7 or 8 days usually beginning about day 145. The initial lambs dropped are usually the sets of multiples, with the single lambs being dropped last.
LBS recommends scanning of the AI group so that multiple bearers can be separated and receive extra feed approaching and during lambing. The survival of twins and triplets depends on milk supply and birth weights, factors related to feeding.
The level of assistance during the lambing needs to be considered. Generally, if females are used to a certain level of lambing assistance, maintain it. If the group is not usually assisted, increasing your presence can create more problems than it solves. There is a reasonable ‘storm’ of lambs or kids, and confusion increases if the mob is disturbed during this period. Predator control is essential, particularly if the AI group is lambing prior to general lambing.
At the end of the process we hope you have a good number of high quality progeny. As a minimum we expect clients to mark the same percent of lambs or kids from their AI as they mark from their paddock joining. Most of our clients mark a higher percent than paddock joining, due to the LBS experience, careful routine followed, extra feed and ‘TLC’ the AI group receive.
Compared to most species, controlling the oestrus cycle of does and ewes is very successful. Ewes and does reliably ovulate in the desired time span, hence laparoscopic AI programmes give very good results (60 - 80% conception rates) compared to the average for cattle fixed time inseminations (say 40 - 60%).
Good animal management and careful insemination with good quality semen frequently sees conception rates up to 80 - 90 %, and 100% conception has been achieved. On the converse, with any management problem low results are possible.
LBS prides itself on achieving the maximum conception rate in all circumstances, not in merely doing as many inseminations per day as possible. “Quality not quantity” is the aim.
Embryo Transfer in Sheep and Goats
- Donors might average 5 to 12 good embryos per flush
- Donors can be reflushed every 2 months
- Good embryos should become pregnancies 60% to 80% of the time
- Freezing of embryos permits sale and implanting at any time.
Embryo transfer (ET) is a useful tool to achieve more progeny f
rom one ewe or doe or a group than is possible in the usual lifetime of a female. The technique is also referred to as Multiple Ovulation and Embryo Transfer (MOET).
A simple outline: A series of hormone injections are given to donor females, causing the release of multiple ova (eggs) at a controlled time. Fertilisation of these multiple eggs is achieved by standard laparoscopic insemination perhaps combined with natural service. Six days later the fertilised ova are flushed from the uterus of the donor. They are microscopically examined, graded, and transferred as single or twins to the uteri of synchronised recipient females.
What ewes or does are suitable as donors?
As long as the donor has a healthy reproductive tract, and has achieved puberty, she is a candidate for ET. Young animals, even as young as 8 months old, can be flushed successfully, but it is preferable to see a few natural cycles from a young donor prior to the commencement of a program. There is a greater probability of a small response from very young donors.
Any history of infertility also increases the probability of getting a below average result. Sometimes valuable females are entered into a program for just this reason, but the result can be unpredictable. Sometimes the cause of their infertility precludes fertile embryos ever being recovered.
How many embryos will each donor give each flush?
The range of fertilised, quality embryos recovered per flush is 0- 30. Nil, one and two are very common recovery numbers, but usually this is balanced by one or two big responding donors bringing the average of the group to 5-12.
Some donors will not give viable embryos for the following reasons;
- No response to the hormone treatment. The donor has no, or a very small number of ova released.
- Fertilisation of the released ova failed. Semen quality needs to be high. Despite high quality semen, in some cases, especially large responses, fertilisation does not occur.
- Fertilisation occurred, but the embryos start to die after a few divisions. These are referred to as “degenerate” embryos and are not transferred to recipients.
There is a genetic component in how each breed responds to the hormone given to stimulate multiple egg release (Follicle Stimulating Hormone, or FSH). Some donors do not respond to a standard dose, whilst others have a large response. The response by individuals is quite repeatable; if a donor gives a large number of embryos in one program, she is likely to respond well when re-flushed.
How many pregnancies will result from good embryos?
60 - 80% of good embryos should result in pregnancies, but recipient fertility and management is the key. Higher or lower results can occur according to the level of recipient management. LBS can guide you in the best treatment of recipients to maximise results
Every recipient presented for implantation is laparoscopically inspected to ensure good uterine and ovary health. To ensure a maximum pregnancy result, any recipient looking less than perfect will not have an embryo implanted.
Some embryo programs are designed around the freezing of embryos. This can be for the sale of embryos, export, or storage to implant at a later time.
Sometimes too many embryos are recovered for the available recipients; excess good embryos can be successfully frozen, stored in liquid nitrogen and then implanted months or years later.
Only very good quality embryos survive freezing and thawing. All embryos are damaged to some degree by freezing so this results in a slightly lower (5 - 10%) conception rate than might be achieved if the embryos had been implanted fresh.
How often can a donor be flushed?
Donors are given treatment at flushing to help the uterus and ovaries return to their normal state. After about 2 weeks the program can begin again, so donors could be flushed every 5 to 7 weeks
A group of unselected donors (say 10 or more) should average 5 - 12 good embryos per donor with a 60-80% pregnancy rate in recipients. Results are improved by flushing a group and removing from future programs the poor performers. Generally donors settle into a pattern of good or poor response.
LBS has been performing ET programs for over 25 years, and uses the latest techniques and embryo medias to achieve the maximum result. Above all, LBS places a high priority on a careful surgical technique that will permit multiple flushes and the full future fertility of donor.