19 June 1998

Sorting fresh sperm comes off

Worrying about whether your

best cow will have a heifer

or bull calf could become a

thing of the past, now

insemination with sexed

semen could soon be

commercially available.

Jessica Buss reports

INSEMINATING cows with sexed semen looks set to become a reality for the UK cattle industry following the installation of a US-developed machine at Cogents base in Cheshire a few weeks ago and farm trials due this autumn.

Sexed semen has previously been used for in-vitro (test tube) fertilisation because only a small proportion of sexed semen has been produced and the process has been slow, says Cogent director Tim Heywood.

Sperm sorting has been refined at Colorado State University in the US and means technicians can produce enough accurately sexed semen for insemination, which is then inseminated using a new deep uterine technique.

Now 12-14% of sperm are recognised as male or female and sorted with 90% accuracy, says Cogent vet Arthur Redpath.

The insemination technique has resulted in a 42% conception rate using sexed fresh semen compared with 52% for frozen semen inseminated conventionally in one US trial, he says.

Further development will hopefully allow semen to be frozen so that it can be distributed widely. The first fresh sexed semen was inseminated last week at Cogent.

The deep uterine insemination technique developed by researchers at Colorado State University is similar to the procedure used for non-surgical embryo transfer, adds Mr Heywood. A catheter containing sexed semen in an AI straw, is inserted 12.5-20cm (5-8in) through the cervix and semen is released two-thirds of the way into each uterine horn.

This insemination technique allows a low dose of sperm to be used so each straw of sexed semen contains only 0.3-0.5m sperm, compared with a frozen straw of semen which contains 15-20 million sperm.

"Deep uterine insemination is easier than embryo transfer because insemination is carried out when the cow is on heat and the cervix is open – embryo transfer takes place seven days after heat.

"Farm trials of the UK-produced sexed semen will start this autumn to check the accuracy of sexing and conception rates and allow improvements to the techniques to be developed and tested," says Mr Heywood.

He explains that the initial benefit will be producing heifers from nucleus herd stock to increase the number of potential bull mothers. Sexed semen will also be used for production of embryos, both to study the accuracy of further developments of the technique and for transplant into nucleus herd cows.

"We also hope Cogent members will begin to benefit soon. Members will be able to confidently breed replacements from their maiden heifers, instead of using an easier calving beef sire, because 90% of animals will have a heifer calf, ensuring easy calving and, therefore, improving welfare," he says.

Sexed semen will speed genetic progress of members herds because almost all replacements will be bred from the youngest highest merit stock in the herd rather than second to fifth lactation cows. Except for a few top cows, which could also be used to breed heifers, the rest of the herd can be served with beef semen which could also be sexed to produce higher value male calves.

Mr Heywood believes that the cost of using sexed semen will be significantly outweighed by the benefits gained from using it.

The machine producees 1000 sexed sperms a second at 90% accuracy, but will get faster.

Now 12-14% of sperm are recognised as male or female, says Arthur Redpath (left). But setting up the cytometer can take hours, according to operator Innes Drummond.

How does it happen?

Fluorescent dye

A fresh semen sample for splitting is first treated with a fluorescent dye and allowed to incubate so dye is taken into the sperm, explains Cogent vet Arthur Redpath.

The fluorescent dye is attracted to DNA in the sperm and because on average X chromosomes which will produce a female embryo contain 3.8% more DNA, they take in more dye.

Dilution

The sample is then diluted using different solutions to buffer and protect it through further processing. It is then ready to be sorted through the Cytometer.

Sorting

To be sorted, the sample must be passed through the machine slowly enough for it to measure the dye in each sperm, so it is made into a stream of tiny droplets by passing it through a funnel. Liquid is also run into the funnel to protect the sperm which are injected slowly into its centre.

Each third or fourth droplet running through the machine will contain a sperm, so helping to ensure there are never two sperms in a droplet.

Investigation

"Each droplet is investigated by a laser and two detectors to identify how brightly the sperm fluoresces. The computer analyses the fluorescence, and initiates a positive or negative electrical charge on the sperm it identifies as containing a male or female producing chromosome to allow separation.

"The computer has to decide to electrically charge the sperm identified, and by that time another 30 to 40 droplets have been investigated. These are then separated into sperms with a positive, negative or no charge using a magnetic field.

Speed

"The machine investigates 6000 droplets per second, giving us about 1000 sperms a second of the desired sex," says Mr Redpath. This is faster than previous processes, however the machine could produce 80,000 drops per second, running through sperm at about 60mph, and Cogent and Colorado State University staff hope to increase the speed of operation after fine tuning.

Splitting

The sexed sperm is then split into different test tubes that containing liquid to soften their landing.

After splitting, the semen is concentrated, rediluted and loaded into straws for insemination.

Difficulties

Setting the cytometer up is difficult, says operator Innes Drummond. Aligning the stream of semen with the lasers and detectors can take hours to fine tune and set up after the machine is stopped, especially when air gets into the machine.