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Sheep breeding gains from frozen sperm technology.

Sheep Breeding Gains From Frozen Sperm Technology

Their long tails quivering furiously, thousands of tiny sperm cells swim across a gray screen. Fernando Rodriguez leans forward slightly, staring intently at the black and white TV in front of him, where the sperm are flagellating.

The screen projects the magnified view of a drop of ram semen, seen under his microscope.

"I give this one a 3.5," says Rodriguez, carefully noting the number in his notebook. "That's out of a possible 4--the top score."

Unlike an Olympic judge, he's not interested in form or style of the tiny swimmers. Instead, he's looking at how many of the sperm can still move their tails after being thawed from a deep freeze. The score--commonly referred to as percent motility--estimates the percentage that are still alive.

Rodriguez, an animal scientist at the ARS Range Sheep Production Efficiency Research Unit in Dubois, Idaho, studies how gel from the common aloe vera plant can help sheep sperm survive a stint in liquid nitrogen. At -- 196 [degrees] C, liquid nitrogen holds sperm cells in suspended animation until they're needed at a later date.

It's all part of a new scheme to help make artificial insemination (AI) feasible for sheep.

Freezing and storing of semen and AI are already widely used by dairy and some beef cattle producers. The practices enable them to save sperm from superior animals with desirable genes for a timed insemination with good breeding animals. Ranchers can thus replicate traits from superior animals in other herds and selectively breed their own flocks to boost desirable characteristics.

The procedure also helps prevent diseases that are spread through sexual contact.

For sheep ranchers, the technique could cut the number of rams needed for breeding on the farm.

"Ultimately, we hope to boost lamb production, which could make lamb more plentiful and less expensive for consumers," says Rodriguez.

However, AI isn't as easy in sheep as it is in cattle. Ram spermatozoa don't survive freezing as readily as those from cattle, possibly because the cell membrane isn't as rigid. And a ewe's cervix (the narrow outer end of the uterus) has folds that form blind, dead-end loops, an arduous trip for even the most hardy sperm.

Research at the Dubois lab addresses both problems--by taking special care to protect the sperm during freezing and by placing the semen directly in the ewe's uterus with a simple surgical procedure.

Rodriguez experiments with semen diluents--substances added to the semen before the deep-freezing process--and other tactics to enhance the sperm's survivability.

He's found that the pulpy gel from the aloe vera plant--long touted for its healing properties--makes an ideal diluent component in extenders for semen. Because ram semen is so concentrated (between 1 and 3 billion sheep semen per milliliter, compared to 800 to 1200 million in bulls), the researchers dilute it before freezing, so it can be used for several insemination doses.

Rodriguez first hit on the idea of using aloe vera while he was a graduate student at Sul Ross State University in Alpine, Texas. One of his professors had found that aloe vera offered promise for treating certain vaginal and uterine infections in cows. But a shortage of infected cows prevented further studies to test the theory. Meanwhile, Rodriguez noticed that cattle sperm mixed well with the gel, so he began testing it as a diluent for freezing semen.

The goal, says Rodriguez, was to find an extender to use in developing countries. "Aloe vera grows naturally in 75 percent of lesser developed countries," he notes.

He later entered a doctoral program at New Mexico State University and switched from cattle to sheep. A nursery in Texas donated several aloe plants, which Rodriguez cultivated on campus for 2 years during the study.

After harvesting, he split the thick, fleshy leaves down the middle--"it's sort of like skinning a fish"--and collected the clear, gooey gel from the plants.

"Aloe vera gel contains glucose, fructose, and other plant sugars that may serve as an energy source for the sperm," says Rodriguez. "Because the gel is so viscous, it seems to coat and protect the cells during freezing."

Researchers elsewhere have experimented with other unusual extenders, such as coconut milk or tomato juice, which also contain plant sugars. While they preserve semen at room temperature for a time, none work as well as aloe vera gel for freeze protection, says Rodriguez.

Other extender additives included with the aloe are egg yolk, which protects sperm during the cool-down period before freezing, and glycerol. After months of refining the ratios of the extenders, Rodriguez found that a diluent made of 40 percent aloe vera "works very well." The rest of the diluent, about 50 percent, is sodium citrate, a common buffer solution.

Rodriguez also adapted a pellet-freezing technique for the semen that's used for cattle semen in some parts of the world.

After mixing in the diluents, he hoists a large block of dry ice onto the lab bench. He presses the end of a small, blunt metal rod into the block to form button-sized indentations in the ice. Next he places 3 to 5 drops of the semen mixture into the holes, where it quickly freezes, forming small, round pellets. These are scooped up and placed in plastic goblets, which are then lowered into a tank of liquid nitrogen.

Rodriguez tests the sperm's progressive motility--gauged by the score of 0 to 4--before freezing and after thawing.

"An excellent sample usually has between 50 and 65 percent of healthy sperm cells after thawing," says Rodriguez.

Many semen samples, however, have considerably fewer survivors--between 20 and 50 percent, depending on breeds, rams, and time of year when the semen was collected.

One possible explanation for some semen's poor freezability relates to levels of naturally occurring chemicals, called polyamines (PA's). Several studies from other labs suggest that one PA, spermine, is important for keeping other cell membranes stable. Low levels of PA's would likely contribute to a poor, post-thaw survival, Rodriguez reasoned.

Rodriguez hopes to learn whether boosting polyamine levels will enhance freezability. Initial results show that washing off the seminal plasma from the sample and adding spermine to the diluent appears to help frozen-thawed sperm swim farther and faster.

Researchers determine those values with the bovine cervical mucus test, which measures the distance sperm can travel during a set time (90 minutes, in this case) through a tube of the mucus. The procedure is also used in clinical medicine to study human fertility.

Helpful as it is, the cervical mucus test is no more than a model. The ultimate objective is to identify sperm that successfully impregnate ewes.

To that end, the Dubois scientists have adopted a technique known as laparoscopic AI that allows AI technicians to see inside a body. Laparoscopes are narrow, flexible or rigid tubes equipped with optics and a light source that allow veterinary surgeons to see inside a body. In surgery on humans, similar instruments are used to simplify invasive procedures.

"By inseminating ewes this way, we not only bypass the cervix, we also avoid having to take the ram's changeable sex drive into account," says John N. Stellflug, an animal physiologist who performs the techniques at the Dubois station. During the summer months, for example, the ram's libido tends to wane and natural breeding becomes less efficient.

The scientists treat the ewes with hormones to stimulate ovulation--the release of an egg from the ovaries. Just before the AI procedure, Stellflug gives the sheep a mild tranquilizer.

He places the ewe on her back in a cradle to expose her abdomen. After applying a local anesthesia, Stellflug makes a small incision--about the width of a little finger--to insert the laparoscope.

"If we just want to check whether a certain ewe is ready to be bred, we use the laparoscope to visualize the uterus, the ovaries, and the corpus luteum--which forms when the ovarian follicle ruptures to release an egg," says Stellflug. "That tells us we have a reproductively active ewe."

But he doesn't actually look at the ovaries during the AI procedure. That's to avoid interfering with ovulation, which could occur after insemination.

Stellflug pokes a tiny needle in the uterus and delivers a few drops of semen--containing about 25 million. sperm--to each of the two uterine horns.

The entire procedure lasts under 3 minutes. Afterwards, Stellflug sprays the tiny cut with an antibiotic. No stitches are needed. He gently shifts the ewe from the cradle to her feet, and she trundles off to rejoin the flock.

"We've inseminated over 3,000 ewes with frozen, then thawed semen in the past 2 years," says Stellflug. "The proportion of successful pregnancies from these artificially inseminated sheep is close to 70 percent. That's similar to that of natural mating during the mature ewes' first heat--or estrus cycle--in the breeding season."

Although the cost of laparoscopic AI makes it impractical for the average sheep producer to use at this time, researchers find it a valuable tool.

"Perfecting AI in sheep could help U.S. sheep producers in several ways," says Rodriguez. "We could more easily use frozen semen from breeds around the world to incorporate desirable genes into our flocks here. Also, by saving semen from their best rams, ranchers could improve their own flocks."

A few producers in the United States are using AI to introduce genes from foreign breeds like the Booroola Merino from Australia. Booroola ewes carry a dominant gene for high ovulation rates. Introducing their genes into flocks is a possible way to boost production, says Stellflug. Other Merino varieties from Australia are known for their fine, soft wool.

Fernando Rodriguez and John N. Stellflug are at the USDA-ARS Range Sheep Production Efficiency Research Unit, U.S. Sheep Experiment Station, Dubois, ID, 83423. Phone (208) 374-5306.

PHOTO : Research flock at the U.S. Sheep Experiment Station near Dubois, Idaho. (K-4166-5)

PHOTO : Animal scientist Fernando Rodriguez checks survival of ram spermatozoa that have been frozen in liquid nitrogen, then thawed. (K-4163-6)

PHOTO : Pellets of ram semen frozen on dry ice. (K-4165-10)

PHOTO : Technician Bill Gardner stores semen pellets in liquid nitrogen. (K-4162-1)
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Author:Corliss, Julie
Publication:Agricultural Research
Date:Aug 1, 1991
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