Simon de Graaf
Status of sexed ram semen in 2022
Probably the most common question I am asked by stud breeders and sheep artificial breeding companies is some version of "where are we at with sexed ram semen?", quickly followed by "does it work?", "do I have to send the rams to the sorting facility?", "how much does it cost?", and "is frozen semen available?". Short answers to these questions are 'yes', 'yes', 'it depends', and 'sort of', but the long answer is a little more complicated so let's explore the issues a bit more below. Alternatively, if you're short of time, jump to the summary.
What is sexed semen?
Ram sperm carry either an X or Y chromsome, which along with the autosomal chromsomes are delivered to the ewe's egg within her oviduct at the moment of fertilisation. If an X sperm penetrates the egg then you get a ewe lamb. If a Y sperm penetrates the egg you get a ram lamb. In a normal ejaculate, X and Y sperm exist in a roughly 50:50 ratio, hence in natural joining systems and in AI programs with conventional (i.e. non sexed) semen you end up with roughly 50% rams and 50% ewes in your lamb drop.
Sexed semen contains a different ratio of X:Y bearing sperm, made possible by the magic of modified flow cytometry which can separate X and Y bearing sperm based on their DNA content. STgenetics are the only company in the world that produces sexed ram semen and they use this method, which is further detailed below. If you come across sexing 'agents' or 'additives' sold online that can simply be mixed with an ejaculate to produce sexed sperm, don't buy them - they don't work!
How does the sexing process work?
As I mentioned above, sexed semen is enriched in sperm of one sex or the other using a process known as modified flow cytometry. This process involves sperm from the ejaculate being labelled with a special fluorsecent dye called Hoechst 33342, which binds to the DNA within the sperm head. The X chromsome of the sheep contains 4.2% more DNA than the Y chromosome, so those sperm containing an X chromsome are labelled with slightly more Hoeschst 33342 than the Y-chromosome bearing sperm. It is this measurable difference in DNA content between the X and Y sperm which is then exploited by a flow cytometer, such as the latest model, the Genesis III pictured below.
Genesis III modified flow cytometer developed by Cytonome and STgenetics for sperm sorting. The Genesis III is the latest generation of sperm sorters and contains 3 heads. Three Genesis III machines i.e. 9 heads can sort ~250 million sperm per hour. Photo supplied by KM Evans (dec) and Vish Vishwanath (dec) of ST Genetics.
The details of how this machine works are beyond the scope of this post (for more info feel free to read my PhD thesis), but the short version is that stained sperm pass through this machine in single file, each encased in a single droplet of fluid and are identified as X if they fluoresce very brightly (as they contain more stain) and Y if they fluoresce less brightly. Once identified as X or Y sperm, the droplet they are travelling in is given a postiive or negative electrical charge, which allows them to be drafted one way or another as they pass through electrostatic gates. X sperm one way, Y sperm the other way, with collection into separate tubes. The 'gates' set around the X and Y populations determines how pure each population is, but in order to balance purity with speed of sorting, it is usually set around 90%. In commercial practice, usually only one sex is collected at a time, so if the client is after 'female' sexed semen then only droplets containing an X sperm are charged and collected, the rest remain uncharged and are disposed of as waste. An example of what this looked like on the software running a modified flow cytometer circa 2014 is shown below.
Each sperm or 'event' detected by the modified flow cytometer shows up as a dot, with it's location dependent on it's level of fluorescence. Sperm with higher levels of fluoresence carry X chromosomes and in this example have a box drawn around them or are 'gated' to tell the machine to charge the droplet they are contained within for separation from the rest of the droplet stream, and subsequent collection as 'female sexed semen'. Image supplied by Vish Vishwanath (dec) of ST Genetics.
Post-sorting, sperm must be re-concentrated so they are centrifgued (literally 'spun down'), the supernatant removed, extender added for liquid or frozen storage, before packaging into straws (for the most part) and either sent for immediate AI or frozen in liquid nitrogen.
How does the sexing process affect the sperm?
The entire process of sperm sexing is technically and biologically very demanding. Sperm are exposed to a multitude of stressors through the sexing process and also afterwards during reconcentration and preservation. Ram sperm deal relatively well with these stressors, or at the very least, processes have been developed over many decades of research to minimise their effects, and as a result fertility of sexed ram semen is generally quite good even at low AI doses. Low dose AI is necessary due to the time consuming process of sorting. In cattle, conventional AI doses have over 10 million sperm cells, while sexed doses originally contained 2.1 and more recently 4 million cells. The combination of low sperm numbers and ‘stressed’ sperm has meant that for cattle, fertility of sexed semen was around 70-80% that of conventional semen, although that has improved in recent years thanks to increasing sperm numbers per dose and changes to media and other processing steps known as ‘SexedUltra’. For sheep, research at The University of Sydney in the early-mid 2000s developed and refined the technology to a point whereby the fertility of sexed frozen-thawed ram sperm cells was similar to conventional frozen-thawed AI dose controls (1 million motile sexed equal to >25 million motile non-sexed, de Graaf et al. 2007), but translation of these findings from a highly bespoke research setting to practice in a commercial sexing laboratory has proven a little more difficult.
In a commercial sorting facility, the needs of the sperm cells must be balanced with the financial realities of operating a profitable business. As a result, batch processing and use of media in some processing steps originally developed for bull sperm likely reduce the quality and function of the resultant sexed ram sperm cells in comparison to when they are produced in a research setting. Nevertheless, after additional development by STgenetics and The University of Sydney in the late 2010s (funded by Australian Wool Innovation, checkout a podcast on this project here), good fertility was obtained with sexed liquid-stored sperm (sexed dose with 10M total sperm = 65% pregnancy rate, conventional non-sexed dose with 25M motile sperm =78%), accuracy of sexing was >90%, and so sexed ram semen was launched as a commercial product in the USA in late 2018, followed by the UK/Europe and then South Africa and Australia/NZ in the year or two after. Russia also apparently had a sorting facilty dedicated to production of sexed sperm from small ruminants, but the war in Ukraine has meant this lab is no longer operating.
What's the fertility of sexed ram semen produced in 2022?
Since its commercial launch, the vast majority of inseminations with commercial sexed ram semen have been carried out with semen which has been collected fresh on the site (or within an hour or two) of the sorting facility at Total Livestock Genetics in Camperdown Victoria and then sent as a liquid product at room temperature for subsequent AI within 24h. These are important logistical considerations as rams must be sent to the sorting facility for collection and the AI itself must be at a location where semen can be transported within approximately 12h following the completion of processing. The actual number of cells within a dose of sexed ram semen are at the discretion of the customer following discussion with STgenetics, but usually range from 2-6 million total cells (cost varies accordingly). Samples are tested for motility, concentration and acrosome integrity prior to leaving the sorting facility with expectations that the vast majority should still have their acrosomes and over 70% should still be motile. Motility should be similar when samples arrive on site for AI.
Fertility results from the use of this sexed liquid-stored ram semen has varied greatly, although efficacy of the sexing process is always high (~90%+ lambs born of the predicted sex). Some programs have been highly successful with pregnancy rates akin to those obtained with frozen conventional semen in the same program (usually around 70%), but other reports of ‘failed programs’ also exist with results of 10-20% pregnancies or lower. The likely cause of this variation is two-fold:
Most sexed semen programs in sheep currently only utilise a single sire. Use of a single male increases the risk a program could fall victim to male factor infertility (or subfertility), but this risk is amplified by the low dose of sperm in each inseminate. Poor fertility in these circumstances may be less to do with the sexing process and more to do with the fact that particular sire has an issue when AI dose drops below the recommended standard. It is well known that variation in fertility between males increases as AI dose decreases. To ameliorate this risk, it is recommended that multiple sires be used in the AI program if possible. It would also be useful for research to be conducted to ascertain the true minimum effective AI dose of liquid sexed ram semen and lab production then remain at that level, rather than offer the range of doses as is current practice.
There is huge variation in synchronisation method and AI time in the sexed ram semen programs conducted across the globe. In some cases, additional synchronisation hormones introduced into programs in an effort to improve synchrony and fertility may in fact be doing the opposite e.g. prostaglandins, GnRH agonists given at the wrong time in the cycle. Further, AI too early or late with respect to ovulation may have a magnified negative impact on fertility when compared with a conventional AI dose. Research is needed to establish how sensitive low dose sexed semen doses are to AI timing, but it stands to reason that the best time for insemination is in the hour or two prior to ovulation, which in most systems utilising CIDRs+400iu eCG would be around 55h post CIDR removal. i.e. aim to AI 53-55h post CIDR pull if possible, 51-55h if a large program. Use the synchronisation protocol that has worked well on your property in the past.
Encouragingly, despite tales of high variation in the 20/21 season, anecdotal reports from the 21/22 breeding season would suggest most sexed semen programs went quite well and results were indeed comparable with the fertility obtained for other non-sexed semen used in the stud's wider AI program conducted at the same time. This may be a function of additional research development by STgenetics in the last 12 months to finesse their processes as well as improved transport logistics and experience of all parties involved in these programs.
What about sexed frozen ram sperm? Can I use sexed ram semen in ET programs?
To date, limited inseminations have occurred with commercially produced sexed frozen-thawed ram semen due to very low fertility (<20% pregnancy rate). STgenetics have recently reported the processing methods for this product have improved, similar to the SexedUltra process applied to cattle, but publicly available fertility data is scant. While it is now possible to purchase frozen-thawed sexed ram semen, care should be taken in its use until large scale fertility data is available with this sperm type. Likewise, use of the current liquid or frozen sexed ram semen products in superovulated donors is unproven and the risk of low fertilisation rates is high, especially if only a single dose of 2-6 million total sperm is used. We have good data from this mid-2000s that the research version of sexed ram semen (even when frozen) at doses of 15M motile sperm worked quite well to fertilise superovulated donors (de Graaf et al. 2007) so if you are intent on using sexed sperm in an ET program, perhaps consider inseminating a couple of straws per donor until there is more data on the likelihood of success at much lower AI doses.
How much does it cost?
It depends on the number of doses you want to order and how many sperm you want in each dose. To answer this question you'll need to contact TLG and STgenetics as per the below. Ballpark, per dose, less than the average price of a straw of ram semen plus agistment and transport costs to/from Camperdown for your sires.
I want to use sexed semen in my next AI program. How do I organise this?
Talk to your current artificial breeding company. They'll probably already know the process and can organise everything for you. Here's a full list of all sheep artificial breeding companies in Australia if you are looking for a new provider.
Alternatively, if you're a sheep artificial breeding company wanting to organise this for your clients or a producer keen to do some of the legwork yourself then contact Ruth Barber at Total Livestock Genetics (email@example.com; 03 5593 2016) for small ruminant sexed semen processing and to discuss options regarding agistment of sires on site or at another collection centre close to TLG/STgenetics. TLG will then contact STgenetics to confirm availability, customer requirements, semen production logistics and any other details. Neel Ganesan at STgenetics (firstname.lastname@example.org) can be contacted directly should you have any queries related to the sexed semen product itself.
If you're in the USA, get in touch with Reproduction Specialty Group based in Indiana but who operate nationwide. In the UK/Europe talk to your current AB company to see if they can book you a slot into the STgenetics/Cogent labs in Chester, otherwise talk to AB Europe who can definitely help you out. In South Africa, Ramsem have partnered with STgenetics to offer sexed ram and goat buck semen there.
Summary and some final advice for artificial breeding companies and seed stock producers
To conclude, the commercialisation of sexed ram semen is an exciting development for the artificial breeding of sheep, but the commercial use of this technology remains in its infancy for small ruminants in comparison to cattle. Fertility is generally good with the fresh i.e. liquid-stored product and similar to what you might get with non-sexed sperm, but results haven been quite variable. Sheep (and goat) artificial breeding companies and their clients must be aware of the logistical issues the use of liquid stored sexed semen will cause, and mitigate program risks by recommending use of multiple sires and tight AI timings. While AI dose is not standardised, I would suggest opting for 6 million total sperm despite the added cost, in order to maximise the chances of program success. Although use of sexed frozen semen remains a risky proposition for now until fertility data from controlled field trials is undertaken and made publicly available, the high fertility previously obtained with this sperm type in a research setting is proof that this too will work in time and a good quality sexed frozen product for sheep will no doubt be available in the future.
In the meantime, I encourage you all to consider how sexed semen might be of use in your sheep enterprise and whether incorporation of this long sought after reproductive technology could assist your breeding and production goals. For those of you that do take the plunge, I wish you the best of luck with your programs, and look forward to seeing many tens of thousands of pre-sexed lambs born in the ensuing decades. Every pre-sexed lamb born is a joy to us researchers who spent so many years in the sperm sexing lab in Sydney and anxiously checking the sex of each new arrival on the research farm.
Greater detail on the process of sperm sexing, the early history of its development and the research conducted by Sydney Uni from the early 90s to 2010s to develop the technology in sheep can be found in my PhD thesis (download below) as well as the review articles 'Sperm sexing in sheep and cattle: the exception and the rule' (de Graaf et al. 2009) and 'Biological and practical lessons associated with the use of sexed semen' (de Graaf et al. 2014).
More information on sexed semen can also be gained by listening to the Sexed Semen episode of the Repro Radio podcast where we speak to STgenetics Australia lab manager Neel Ganesan.
Checkout the following links for:
Video resources on the artificial breeding of sheep
Repro Radio podcast episode on the use of 'Reproductive technologies in animal agriculture'
Commercial livestock semen assessment services
More information about Professor Simon de Graaf or to get in touch (email@example.com)