- Collection and use of bovine semen in Australia
- The use of sexed semen in an embryo transfer program
- Embryo transfer for Wagyu cattle
Collection and use of bovine semen in Australia
By Dom Bayard
Collection
The collection and use of bovine semen has been widespread in Australia for a number of decades and since the industry was deregulated, some years ago, the distribution of semen between states has increased dramatically. At the same time, the growth in the number of successful local Japanese Black AI sires has remained low. It is important for breed futures to stimulate this market. The following is an overview of local bovine semen collection, with specific references to the Wagyu industry.
Australian Semen
1. Unlicensed semen
This is semen produced on farm or by taking a sire for a day visit to a central collection centre. This semen is collected and processed without any health tests being done on the sire, and if done on farm, often with an estimated range of sperm per ml and analysis of sperm morphology. This system of semen collection is very popular in Australia and is a good way of protecting genetics as an insurance against bull death or for future “on farm” use.
These straws are frozen in wine red straws and the semen density per straw often varies between sires, depending on how many straws are required and the semen quality judged in its raw state. The disadvantage of this type of collection is the risk of the sire carrying diseases which are transferred through semen, and are not destroyed with the freezing process. There is widespread private distribution of semen between Australian cattle breeders using this low cost method, which is also likely to be ideal for many small Wagyu breeders – after a careful risk analysis.
2. Licensed domestic semen
This semen is produced on an artificial breeding centre. There are a number of centres located throughout Australia, registered and licensed through the Department of Agriculture. Each of these centres undergoes an annual audit and meets a specified set of standards as laid down by the Department of Agriculture. Before a sire enters the AI centre it is required to undergo a number of health tests on the farm of origin before being allowed onto the entry facility of the AI centre. Once the results of the on farm tests are known, the bull arrives on the AI centre in an entry facility where it undergoes another round of testing. Once the results of this round of testing are complete the sire is allowed onto the centre proper where licensed semen can then be collected.
There are a number of advantages to having licensed semen collected. As well as the obvious benefit of knowing the health status of the sire, the centre also collects and processes the semen under controlled laboratory conditions and generally are able to closely look at both sperm density and sperm morphology. The amount of viable sperm per straw is much more accurately controlled under these conditions, and there is generally better quality control over the semen product. Semen from licensed semen is put into a different colour straws to differentiate breeds i.e. green for Jersey, white for Holstein, blue for Ayrshire etc although in recent years this has not been as strictly done as it was in previous decades. In Australia semen is generally processed in ¼ ml straws with between 10 and 20 million sperm per straw except in the case of sexed semen where generally 2 million sexed sorted sperm per straw is the norm.
Licensed semen also has a more rigorous system of straw labelling. Most licensed straws carry much more information than unlicensed straws and include the sire name, the NASIS code (specific bull code issued by the ADHIS for individual national records) a batch or collection date and the name or code of the centre producing the semen.
3. Licensed export qualified semen
This semen is similar to the 2nd type of semen (licensed) but generally there is a higher standard of health testing depending upon the destination of the semen. Each country has its own set of protocols for the importation of bovine semen which must be met and so various blood and preputial tests are required at different times.
When considering the collection of semen for an export market it is of great benefit to have the destination countries worked out before commencing the collection work. Whilst it is possible sometimes to have testing done retrospectively to meet countries requirements, it is far easier to do the correct testing at the time.
4. Storage
It is important to store and thaw semen correctly when using semen in an AI or ET situation. There is plenty of literature available on the correct methods of handling and thawing semen that breeders should make themselves aware of before using semen in a program.
This is especially important when using expensive irreplaceable semen or when using sexed semen with its much lower sperm numbers per straw. Always take note and record batch numbers when thawing as there is variability between batches and recording this will be important if a semen fertility problem arises.
Imported Semen
Another option for semen users in Australia is imported semen. The dairy industry has been an avid user of imported semen, especially from Canada and the USA in early years and from various European countries in later years. All semen entering Australia from overseas is licensed semen and has been subject to the protocols laid down by the Australian Department of Agriculture for the importation of semen into Australia.
Whilst this covers the health status of the semen, it does not infer that there is a standard of viable sperm per straw. Semen quality is a separate issue and is not covered by the import protocols. Semen quality is an issue for the collection centre and its own standards and reputation. Be aware that semen quality does vary and that some sires do not freeze well whilst other sires have very high fertility levels. Because there is often a big trade in straws of semen, it is possible that inconsistent handling and movement of straws of semen through various tanks can cause damage to the viability of the semen. This should be considered when purchasing semen privately from other breeders or when purchasing semen that may be quite old.
Unfortunately, in the case of fullblood Wagyu semen, there are very few overseas herds (outside Japan) based on significantly different genetics to those already available within Australia. Nonetheless, there are occasional exceptional animals produced offshore from the same gene pool and these are worth identifying. The key to the evaluation process will be progeny performance data.
Use of Semen
There are a number of considerations specific to the use of AI in Australian Wagyu production:
- Genetic diversity or effective population size in the Australian fullblood Wagyu (Black or Red) has not been officially measured in the local history of the breed. Regardless, Japanese assessment of the home herd in Japan has revealed startlingly low diversity – ironically often as a consequence of excessive use of a very small group of genetically similar AI sires. Australian diversity is likely to be somewhat lower than that of Japan, and AI use in the breed is dramatically lower – based on registration numbers. Increased semen collection and dissemination should therefore be encouraged.
- However, the potential marketplace for most Australian Wagyu semen producers is quite small, and the relatively high production cost of Licensed Domestic and Export Qualified semen makes this an uneconomic proposition for most producers. In order to create a viable semen market covering the broadest range of genetics available (in either the Japanese Black or Japanese Brown – aka Red Wagyu), the production and promotion of the availability of a wide range of ‘unlicensed semen’ is likely to be the only viable stimulus to the maintenance or increase of genetic diversity in the national herd. Producers should be encouraged to ‘milk’ exceptional sires and promote these genetics, with careful reference to the ‘proof of value’ – superior measured progeny performance.
The use of sexed semen in an embryo transfer program
By Dom Bayard
I am often asked for an opinion on the use of sexed semen when flushing donors and whether it works. Many breeders try using it on one or two donors and this can lead to varying results, with some complete fertilisation failures but also some donors that respond well and give pleasing results.
Other issues around this subject include timing of insemination and placement of semen in the reproductive tract including whether placement deep up the uterine horn is advantageous or not.
We have recently undertaken a program here at Goorambat Wagyu using female sexed semen from our own sire GRSFK0473 and another Wagyu sire. I present these results here as an indication of what can happen and there are some interesting points to come out of it.
We set up 36 full-blood Wagyu donors in 2 groups, flushing 18 each day. I must point out that this was not run as a scientific trial but under commercial flushing conditions with the aim of producing as many female wagyu embryos as possible. Each group was managed under the same conditions and preparation , however, FSH dose rates were specific to the individual donors so they varied slightly, with a total of 8 ml being the highest individual rate and 5 ml being the lowest.
GRSFK0473 semen was presented in straws of 4 million sperm per straw while Bull B was 2 million sperm per straw. The donors were inseminated twice; at roughly 12 – 15 hours and again 24-28 hours after the onset of standing heat. The donors using the bull GRSFK0473 were inseminated with 1 straw of semen each time (total of 8 million sperm) and for those using bull B, 2 straws were used at the first insemination and 1 straw at the 2nd insemination. (total of 6 million sperm).
Overall Results
| EMB GRADE | |||||||
| TOTAL | A | B | C | UNFERT | DEG | ||
| GRSFK0473 | Day 1 | 183 | 89 | 5 | 0 | 44 | 45 |
| 19 | Day 2 | 56 | 24 | 8 | 0 | 2 | 22 |
| TOTAL | 239 | 113 | 13 | 0 | 46 | 67 | |
| AVE | 12.58 | 5.95 | 0.68 | 0.00 | 2.42 | 3.53 | |
| Bull B | Day 1 | 91 | 33 | 23 | 0 | 25 | 10 |
| 17 | Day 2 | 141 | 49 | 21 | 0 | 30 | 41 |
| TOTAL | 232 | 82 | 44 | 0 | 55 | 51 | |
| AVE | 13.65 | 4.82 | 2.59 | 0.00 | 3.24 | 3.00 |
| day 1 | |||||||
| EMB GRADE | |||||||
| SIRE | DONOR | TOTAL | A | B | C | UNFERT | DEG |
| GRSFK0473 | A | 32 | 22 | 2 | 0 | 4 | 4 |
| GRSFK0473 | B | 32 | 2 | 0 | 0 | 20 | 10 |
| GRSFK0473 | C | 8 | 3 | 0 | 0 | 3 | 2 |
| GRSFK0473 | D | 17 | 9 | 0 | 0 | 4 | 4 |
| GRSFK0473 | E | 10 | 4 | 0 | 0 | 6 | 0 |
| GRSFK0473 | F | 11 | 3 | 1 | 0 | 2 | 5 |
| GRSFK0473 | G | 18 | 15 | 0 | 0 | 1 | 2 |
| GRSFK0473 | H | 5 | 2 | 1 | 0 | 0 | 2 |
| GRSFK0473 | I | 17 | 13 | 0 | 0 | 0 | 4 |
| GRSFK0473 | J | 10 | 2 | 1 | 0 | 1 | 6 |
| GRSFK0473 | K | 9 | 2 | 0 | 0 | 2 | 5 |
| GRSFK0473 | L | 2 | 2 | 0 | 0 | 0 | 0 |
| GRSFK0473 | M | 12 | 10 | 0 | 0 | 1 | 1 |
| TOTAL | 183 | 89 | 5 | 0 | 44 | 45 | |
| AVE | 14.08 | 6.85 | 0.38 | 0.00 | 3.38 | 3.46 | |
| Bull B | A | 16 | 10 | 3 | 0 | 0 | 3 |
| Bull B | B | 3 | 2 | 0 | 0 | 1 | 0 |
| Bull B | C | 19 | 8 | 0 | 0 | 11 | 0 |
| Bull B | D | 28 | 7 | 10 | 0 | 4 | 7 |
| Bull B | E | 22 | 3 | 10 | 0 | 9 | 0 |
| Bull B | F | 3 | 3 | 0 | 0 | 0 | 0 |
| TOTAL | 91 | 33 | 23 | 0 | 25 | 10 | |
| AVE | 15.17 | 5.50 | 3.83 | 0.00 | 4.17 | 1.67 |
| DAY 2 | Emb grade | ||||||
| SIRE | DONOR I.D | TOTAL | A | B | C | UNFERT | DEG |
| GRSFK0473 | a | 21 | 5 | 2 | 0 | 1 | 13 |
| GRSFK0473 | b | 9 | 7 | 0 | 0 | 0 | 2 |
| GRSFK0473 | c | 6 | 1 | 0 | 0 | 1 | 4 |
| GRSFK0473 | d | 6 | 5 | 0 | 0 | 0 | 1 |
| GRSFK0473 | e | 10 | 5 | 4 | 0 | 0 | 1 |
| GRSFK0473 | f | 4 | 1 | 2 | 0 | 0 | 1 |
| TOTAL | 56 | 24 | 8 | 0 | 2 | 22 | |
| AVE | 9.33 | 4.00 | 1.33 | 0.00 | 0.33 | 3.67 | |
| Bull B | a | 6 | 3 | 1 | 0 | 1 | 1 |
| Bull B | b | 33 | 0 | 1 | 0 | 15 | 17 |
| Bull B | c | 24 | 7 | 3 | 0 | 9 | 5 |
| Bull B | d | 19 | 13 | 0 | 0 | 2 | 4 |
| Bull B | e | 11 | 4 | 3 | 0 | 0 | 4 |
| Bull B | f | 5 | 3 | 0 | 0 | 2 | 0 |
| Bull B | g | 15 | 7 | 1 | 0 | 1 | 6 |
| Bull B | h | 9 | 5 | 2 | 0 | 0 | 2 |
| Bull B | i | 5 | 3 | 1 | 0 | 0 | 1 |
| Bull B | j | 10 | 3 | 7 | 0 | 0 | 0 |
| Bull B | k | 4 | 1 | 2 | 0 | 0 | 1 |
| TOTAL | 141 | 49 | 21 | 0 | 30 | 41 | |
| AVE | 12.82 | 4.45 | 1.91 | 0.00 | 2.73 | 3.73 |
Insemination technique
2 different inseminators were used. On day 1 all donors were inseminated by inseminator A by placing semen well up the uterine horns of each donor. In the case of the GRSFK0473 donors, ½ a straw was placed deep in each horn on both inseminations. With Bull B, 1 straw of semen was placed deep up each uterine horn on the 1st insemination and ½ straw deep up each uterine horn on the 2nd insemination.
On day 2, Inseminator B inseminated all donors . In all cases, semen was deposited just through the cervix into the uterus. For GRSFK0473, 1 straw per insemination was used. With Bull B, 2 straws at the 1st insemination and 1 straw at the 2nd insemination were used.
Semen was thawed as each donor entered the crush so no more than that required for each donor were thawed at the same time. Thawing technique was the same for both days inseminations.
Discussion
In this instance, it appears that the use of 3 straws with 2 million sperm per straw or 2 straws of 4 million sperm per straw is adequate to obtain satisfactory results with sexed semen in an embryo transfer program. It also appears that the use of deep uterine insemination techniques is not necessary to obtain a satisfactory fertilisation rate.
The preparation of donors under controlled feeding and management conditions allow for the use of sexed semen in an embryo transfer program to manipulate the balance of female and male bias. With the returns available in the current Wagyu market, the ability to get the results as shown in this case makes the use of embryo transfer and sexed semen worthy of serious consideration as part of a Wagyu breeding program.
Embryo transfer for Wagyu cattle
By Dom Bayard
MOET (Multiple Ovulation, Embryo Transfer) is a breeding technology tool that has been an option for cattle breeders since the 1980’s in a commercial form. The basis is the use of a drug called FSH (follicle stimulating hormone), which recruits many of the follicles that are available on the ovary at any particular time, and helps them all to grow at once, rather than just the one follicle that grows during a normal cycle.
MOET can be used on heifers from around the 12 to 14 months of age, on lactating Wagyu cows and also on older dry cows that are not in calf. Lactating cows need to be at least 42 days post calving before undergoing a flush procedure and need to have had at least one post calving heat cycle. It is not necessary to wean the calf off the donor.
RESULTS:
Donors can be flushed every 6 to 8 weeks and from each flush a cow should average about 5 transferable embryos. Heifers generally can be slightly less than the average and some donors average well above this number. Whilst these figures may surprise some, it is often only the good results that you hear, but be assured there are some donors that are poor flushers and may never produce the average number of embryos.
The number of embryos’ collected is affected by many variables.
Firstly, some cows produce large numbers of oocytes (eggs) on their ovary during their natural reproductive cycle. These cows therefore have the natural ability to produce larger numbers of embryos than the cow that has small numbers of oocytes produced. It is often seen that this oocyte production follows cow families. Therefore good embryo producers often come from a dam that is a good embryo producer, and in turn, the progeny often continue this trend. Conversely it occasionally occurs that poor embryo producers continue that trend through their cow family. There are exceptions however as other factors can be responsible for results.
Secondly, time of the year and lactation have an effect. Donors on high protein feed (such as rapidly growing young pasture or clover dominance) along with a peak in lactation are really adverse to reproductive results. We therefore try to slightly modify feeding regimes and work with the owner to map out the best methods of feeding to maximize embryo production. We would suggest that all donors be given a Vitamin A, D and E injection along with Vitamin B12 and multimin about 4 weeks before flushing.
Thirdly recipient condition and ovary quality play a role. Recipients are discussed later in this article.
HOW DOES IT WORK ?
A MOET program takes about 24 days to undertake. It commences with an injection of prostaglandin, then insertion of a CIDR into the potential donor. 5-7 days later the donor commences a series of twice a day FSH injections over 4 days. She is given a PG injection and has the CIDR removed at strategic times over this 4- day period and will have a heat the day after the last FSH injection. At that time she is mated to the preferred sire, using a 2-insemination program. Then 7 days later we undertake the flushing regime.
Flushing a donor animal involves giving it a local anesthetic or epidural, then the placement of a catheter into the uterine horns. This looks similar to the method used for A.I. We then run some flush fluid into the uterine horns to collect the embryos that are there. Both uterine horns are flushed and the fluid and embryos collected in a specially designed filter. At the completion of the flush, which takes between 20 and 30 minutes per donor, the filter is searched under the microscope and the embryos collected into a small dish. The embryos are graded for both quality and stage of development. At this time they can either be transferred into available recipients or cry-preserved (frozen) for later use. Frozen embryos can be kept for many years if required.
At the completion of the flush the donor animal is given a PG and she should have a heat cycle within 5 – 7 days. It is important that she does have a heat quickly after flushing to return her reproductive tract to a normal condition so heat detection post flush is important.
DONORS
There always seems to be conjecture about how much FSH the donor should receive to get the best result. In our experience, the Wagyu breed are very sensitive to FSH. Our standard FSH dose is 6 ml, given over the 4 day injection period. Some of our more sensitive responders are only on a total of 4 ml over the 4 days. Compare this with an Angus or Holstein donor who regularly may be on 14 to 16 ml of FSH over the 4 day period. It is important to consult closely with your attending ET practitioner in order to get dose rates right. The Akaushi are not quite as sensitive and are on slightly higher rates. We very rarely go above 8 ml on any donor.
The other extremely important point to make is that Wagyu donors flush much better in lighter condition. It is difficult to get fat donors to flush well and we try to have our donors in forward store condition at best when flush day arrives.
One other point to consider is that we have seen the occasional anaphylactic reaction in Wagyu donors when using Folltropin. This manifests itself shortly after the injection is given with varying degrees of intensity. Puffy eyes, mucous membranes running and patches of hair “standing up” are good indicators. It is worth watching the donors for 10 minutes after they are given Folltropin to just make sure they are not having this reaction.
RECIPIENTS
Recipient animals are those females that the embryos from the donor are placed into, either in conjunction with the flush day or later when frozen embryos are used.
A recipient program is easier to carry out than the donor program but usually involves the use of a CIDR to synchronize heat cycles. In the case of transferring fresh embryos on the same day as the flush, we require the recipients to be on heat around the same day as the donor. The only thing we do at this stage is note which ones are on heat and the time of heat. They are then left unmated until the following week when the embryos will be transferred into them.
Not every recipient that has a heat will be suitable to have an embryo transferred into it. It can vary from 50% up to 90% usage rate depending upon the condition of the recipients’ ovaries on the day of transfer. Each recipient is palpated and the quality of the ovary response noted. Any recipient that is not suitable is left and not used. Only suitable recipients will have an embryo placed in them.
The pregnancy rate from embryos transferred can vary but a minimum expectation should be 50%. There is a slight variation between fresh and frozen embryos with the frozen rate being up to 10% less than fresh transfers. However the transfer of fresh high quality embryos into good recipients can result in much higher pregnancy rates.
Recipient selection is very important. Commonly, recipient selection is overlooked by breeders, who put up cows they don’t want calves out of themselves, or cows that haven’t got into calf during the previous joining period or are carryover cows. These are generally among the lower fertility group of the herd and their selection as recipients is reducing the chances of getting high pregnancy rates. Ideally, recipients are the middle of the range cows that are not over fat or giving huge production figures and are fed correctly. In dairy herds, 2-year-old cows in their 1st lactation generally do not make good recipients as this group of cows is under the most pressure.
Beef recipients do not have the same levels of production pressure on them but again the use of lower fertility recipients will result in lower pregnancy rates.
Both dairy and beef heifers make good recipients. Wagyu embryos are ideal to use in heifers as the smaller birth size of the majority of Wagyu calves makes them a real option. You do need to keep in mind that calving heifers may not have great levels of antibodies in their colostrum and so vaccinating heifers or substituting colostrum from older cows may be considered appropriate in some circumstances.
We would suggest that recipients be given a Vitamin B12, Vitamin A, D & E injection and also a multimin or equivalent injection prior to the ET program. We also strongly suggest that you look at the use of a pestivirus vaccination program to maximize the potential of your recipients.
GENERAL
We are often asked how many times can we flush a donor. With heifers, the suggestion is to flush them only twice or perhaps 3 times before getting them into calf. With cows you may consider flushing them 3 or 4 times before getting them back into calf. It should be noted that occasionally it might take a little while after the completion of a flushing program to get the donor into calf.
It is important especially when considering MOET for the first time that you go through the whole program with your ET technician prior to program commencement and that you understand what is about to happen. This will ensure you obtain maximum results.
GRS is an embryo export centre at Benalla, in southern Australia and also runs the Goorambat herd of fullblood Wagyu. We have had up to 120 Wagyu donors on centre at any one time and have exported to numerous countries throughout the world. We first started in ET in 1990.
Dominic Bayard
Global Reproduction Solutions.
PH +61 3 5764 1224
Fax +61 3 57 641225
Mob 0427 732 538
www.grsembryos.com.au