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From simple beginnings to genetic engineering.

Good Breeding

From Simple Beginnings to Genetic Engineering

Since the days when mankind first became a herder of animals, the genes of livestock have been rearranged by selective breeding within the herd for desired traits.

From those simple beginnings has grown the modern science of genetics, with a dazzling array of tools to help livestock producers in their selections.

"We now have the expertise to improve the reproductive efficiency of our farm animals and improve existing breeds through the direct transfer of genes from other animal species," notes Harold W. Hawk, an animal physiologist with the Agricultural Research Service's Gene Evaluation and Mapping Laboratory at Beltsville, Maryland.

Such switching or engineering of genes offers a host of benefits: more efficient utilization of feed by farm animals, faster growth, greater resistance to disease, even the ability to produce pharmaceutical products in the milk of dairy animals.

In one project at the Beltsville laboratory, scientists are studying ways to improve the survival rates of cow embryos incubated outside the natural mother for about a week, then placed in a surrogate mother for gestation.

"We have been able to increase fertilization rates of cow eggs from 60 percent to as much as 100 percent and have improved the survival of the embryo from 10 percent to 20 percent," says Hawk. "In essence, we're setting the stage for the efficient genetic engineering of cattle."

However, much of the laboratory's work is done on species other than cattle because, Hawk explains, "cows are expensive to maintain, they reproduce slowly, and they have, at best, two offspring per pregnancy."

By contrast, hogs can produce two litters per year, averaging about 10 piglets per litter.

"And many of the techniques that we learn from our research on pigs and sheep can eventually be used to produce transgenic cattle," he adds.

One such technique is the transfer of a gene or gene replica into a newly fertilized egg.

Within a few hours after fertilization, the genetic material of the sperm and egg are naturally enclosed in membranes within the egg. These bodies are called the pronuclei. It is at this stage, before the pronuclei unite to form an embryo, that gene transfer usually takes place.

To accomplish this, the egg is held by gentle suction from a glass tube. While looking through a microscope, the scientist uses a micromanipulator to maneuver a fine glass needle containing copies of the new gene. The egg membranes and membrane of one pronucleus are pierced, and the gene replicas are placed inside the pronucleus.

Embryos of pigs and sheep are then placed immediately into a surrogate mother, but cow eggs are incubated for 7 to 8 days before transfer. A small percentage of these embryos will grow into offspring in which the new gene is "expressed" or working.

Farmers have long dreamed of being able to select in advance the sex of the offspring of their livestock. Generally, beef producers would prefer more males, because steers grow faster than females.

"We are getting closer to that goal," says Lawrence A. Johnson who is at the Germplasm and Gamete Physiology Laboratory at Beltsville. "In the future, sex preselection will be a common facet of livestock production."

Johnson has developed a system for sorting batches of sperm cells based on the amount of DNA they carry.

"Sperm cells carrying the Y chromosome produce males, while sperm carrying the X chromosome produce females," explains Johnson. "The X-bearing sperm carry more DNA, which can be measured using a fluorescent dye and a laser."

The sperm cells are first treated with a fluorescent dye, then passed into a cell sorter, where they flow single-file past a laser beam. The X sperm give off more fluorescent light than Y sperm because of their greater DNA content. Based on the light they emit, the X and Y sperm are collected in separate tubes.

"To date, we have achieved live births with artificial insemination of sorted X and Y sperm of pigs and rabbits," notes Johnson. "At least 75 percent of the offspring have been of the predicted sex."

The same principle would apply in sorting cattle and sheep sperm. However, the cell-sorting procedure can currently sort only about 2 million sperm a day, while 10 to 15 million sperm are needed for conventional artificial insemination of a cow.

An alternative procedure to the use of millions of sperm--fertilization of cow eggs outside the uterus--would drastically reduce the number of sperm needed.

"We are also trying to identify some sort of sex-specific marker on the surface of the sperm," says Johnson. "If we are successful, we may be able to develop a batch procedure to be used in preselecting populations of male versus female sperm and thus make the procedure practical."

Body Fat Affects Puberty

Just as the health and diet of a human mother-to-be can affect her unborn child, nutrition and body condition play a crucial role in producing a calf, says animal scientist Andrew C. Hammond.

Hammond, research leader at the ARS Subtropical Agricultural Research Station at Brooksville, Florida, is working with reproductive physiologist Chad C. Chase, Jr., on how body fat figures into when a heifer reaches puberty and can be bred.

"Puberty is important because one of our biggest problems is reproductive efficiency," Hammond says. "The cattle necessary for a subtropical environment such as we have here in Florida are typically older at puberty and have a longer gestation."

Cattle of the Bos indicus type, as typified by the Brahman breed, are better equipped by nature to survive sultry temperatures. But 4 years of data from Brooksville show the average age of Brahman heifers at puberty is about 602 days, compared with 503 days for Angus heifers, a Bos taurus animal.

Additionally, Brahman cows have a mean gestation length of 293 days, compared with the Angus' 278 days.

"A longer gestation period means the cattle producer has a shorter amount of time in the calendar year in which to get that cow bred again," notes Hammond. "A certain amount of time has to pass after calving before a cow comes back into heat.

"We know that a cow needs to be in a certain body condition to rebreed," he points out. "Quite a bit of work has already been completed on characterizing age and body weight at puberty; we're interested in the heifer's body fat."

To this end, Hammond and colleagues at Brooksville began a 3-year study in the fall of 1990 involving heifers of the Hereford, Senepol, Angus, and Brahman breeds, plus crossbred heifers from Hereford and Senepol parents.

Once the animals are weaned, they're put on one of two diets offering different energy levels. The heifers are then bred to Angus bulls under careful supervision so researchers can note the female's body composition at the time she conceives.

"The practical aspect of all of this is refinement of feeding," says Hammond. "We're trying to determine differences in cattle types and how you might have to feed various types for optimum reproductive performance."

Animal physiologist Robert B. Staigmiller is quick to second the idea of the importance of good nutrition in reproduction.

"You can probably change the age at which the animal reaches puberty by as much as 8 weeks with the right nutritional program," says Staigmiller, who is based at the ARS Range and Livestock Research Unit at Miles City, Montana.

"Nutritional needs are different for different biological types of animals. You need to know your breed characteristics."

But reaching puberty doesn't always equate with immediate pregnancy, he adds.

"We've found that heifers bred on their third estrus cycle have a 21 percent greater conception rate than heifers bred right at puberty," Staigmiller notes.

"We've reasoned there are at least two possible causes for this. Maybe that first egg produced doesn't have as strong a possibility of becoming an embryo.

"The first egg ovulated will not have been exposed to all the hormonal signals of a regular estrus cycle like eggs ovulated later. Or perhaps the uterine environment isn't quite right in a heifer at puberty."

To test this theory of egg viability, Staigmiller and coworkers are taking eggs produced by mature cows and transferring them into heifers at the young females' first or third estrus cycles. Staigmiller says another year of data is needed before the results can be evaluated.

On the question of uterine environment, "We know the tissues of the oviducts and uterus undergo dramatic changes at puberty," says Staigmiller. "These changes may not be completed during the first estrus. This could have an adverse effect on the ability of fertilization to occur, or it could be detrimental to the early life of the embryo.

"Overall, what we've seen so far tells us that it's not sufficient to barely have a heifer at puberty at breeding time," Staigmiller concludes. "A young female's chance of becoming pregnant early is greater if she has reached puberty in time to have two or three estrus cycles before the start of the breeding season."

Twinning Project

A small percentage of the time--about 1 percent for Hereford and Angus to 4 percent for Holsteins--the producer is rewarded with twin calves. According to Keith E. Gregory, an animal geneticist at the Roman L. Hruska U.S. Meat Animal Research Center (MARC) at Clay Center, Nebraska, it is possible, to improve those odds--possible, but not simple.

"We've had a research project on twinning in beef cattle since 1981," says Gregory. "Research has shown that for intensive production systems, twinning has the potential to increase efficiency of beef production by 25 to 30 percent."

Gregory and colleagues at Clay Center began the twinning project with carefully selected foundation females that had naturally produced twins at a high frequency. Their daughters in turn twinned at a rate of about 9 percent. Later, these daughters were fertilized with semen of bulls from Sweden or Norway whose daughters had produced twins at a frequency of about 10 percent.

In their search for sires to use in boosting twinning rates, the scientists consider ovulation rate, determined by rectal palpation, on seven daughters of each young sire for seven estrus cycles. This information not only enables the researchers to pinpoint the best sires, but also helps them select replacement females.

"The twinning frequency since this procedure began has been increasing at about 2 percent a year for the last 5 years," Gregory notes. "In the spring of 1991, it was 23 percent."

But such victories don't come easily, Gregory adds.

"Increasing twinning in a herd has a very high input requirement in terms of management," he emphasizes. "And we think that for it to be really economically viable for a producer, a twinning rate in the range of 40 to 45 percent is necessary.

"Based on the progress to date, we're of the opinion we can achieve that. But we think that superior breeding stock is an essential part of a twinning technology package."

Twins can be a mixed blessing, Gregory warns. "Death loss of twin calves is higher, and we're finding that a cow that's had twins has a longer interval before she can be bred again," he says.

"That's because her body cavity is so full of fetuses that there literally isn't room for her to eat enough of a normal low-energy beef cow diet to maintain herself in good enough condition to rebreed. So you must have these cows on a higher energy diet in their last trimester of pregnancy."

According to Sherrill E. Echternkamp, an animal physiologist at Clay Center, the increased energy requirement and the need for more assistance in calving make it vital that the producer know which cows are carrying twins.

"In twins, there's fairly high calving difficulty," he notes. "They get tangled up together. About 35 percent of twin births require assistance, compared with about 15-20 percent of single births."

For that reason, the Clay Center scientists are increasing their use of ultrasound diagnosis to determine the fetal number, Echternkamp says.

"We're about 85 percent accurate when we evaluate the cows 45 to 85 days after breeding," he says. "Since we've started doing that, we've increased calf survival about 10 percent."

Uterine Blood Flow

In one of the more curious twists of science, Clay Center researcher Calvin L. Ferrell has shown that the uterus itself is much more than just "rented space" for the developing calf.

In a study begun in 1989, purebred Charolais and Brahman cows were injected with hormones to increase the number of eggs they produced. The eggs were fertilized by artificial insemination with semen from purebred Charalois or Brahman bulls.

Then Ferrell and coworkers implanted the purebred Brahman embryos in Charolais cows, and the purebred Charolais embryos in Brahman mothers. Some of the fetuses were recovered for study at 230 days of gestation and others at 270 days.

"At 230 days, the cow hadn't had any influence on calf growth," says Ferrell. "The Charolais calves, regardless of which breed of mother they were in, were nearly twice the weight of the Brahmans--23 kilograms versus 13 kilograms.

"But at 270 days, the Charolais calves that had been inside Charolais mothers weighed about 13 kilograms more than the Charolais calves inside Brahman mothers. And the Brahman calves inside Charolais mothers weighed about 5 kilograms more than the Brahmans inside Brahmans."

Ferrell contends that uterine blood flow may be contributing to the difference. By monitoring the mothers' blood flow to the fetuses through the use of markers infused through catheters in the mothers' veins, the researchers discovered a much lower uterine blood flow in the Brahman cows.

"Also, the placental tissue is smaller in the Brahman--apparently a common trait in Bos indicus cattle," says Ferrell. "That smaller tissue is just not adequate to supply all the nutrients a larger fetus needs.

"If we could control uterine blood flow, we might be able to restrict or improve fetal growth. Presumably uterine blood flow is a genetically controlled trait. If we could find the genes, we might be able to modify or use them as a selection tool."

Bigger calves can translate to more calving difficulty, a hot topic with ARS reproductive physiologist Robert A. Bellows at Miles City, Montana.

"I've been working on causes of calving difficulty, called dystocia, since 1962," says Bellows. "I'd estimate this one problem results in an income loss of more than $800 million annually to U.S. cattle producers."

One common cause of dystocia is a calf that's too big to go through the mother's birth canal. Another problem is abnormal hormone changes in the dam just before calving.

The scientists have noted a difference in precalving levels of the hormones estrogen and progesterone in cows with calving difficulty. These and other hormones play a role in the strength of the cow's contractions and in preparing the birth canal for delivery of the calf, says Bellows.

The stakes are higher than just one calf one year, Bellows adds.

"Cows in labor for long periods of time have poor reproductive performance in the subsequent breeding season, resulting in a reduced or delayed calf crop the next year," he points out.

Cutting back on the pregnant cow's nutrition to try to trim calf size isn't effective, Bellows says.

"We weren't able to accomplish that, but the reduced nutrition did result in poor rebreeding of the cow," he says. "And cutting calf size too much, no matter how you do it, can backfire because very light calves don't gain weight as rapidly or weigh as much at weaning."

Current studies with five breeds of cattle have uncovered differences in the degree to which the mothers will let the calves' genetic potential develop in the uterus.

In the study, Charolais and Shorthorn cows produced the heaviest calves, regardless of whether the sire was a high-or moderate-growth animal, says Bellows. At the other end of the spectrum, Brahman dams repressed fetal growth rate to a certain extent.

Jersey cows, by contrast, produced low-birth-weight calves from low-growth sires and high-birth-weight calves from high-growth sires. Calves from Longhorn mothers were medium-size, regardless of the sire's growth rate.

PHOTO : Hereford twins produced at the ARS Range and Livestock Research Unit at Miles City, Montana. (K-4323-18)

PHOTO : Using compounds in blood samples from heifers, animal scientist Andrew Hammond monitors nutritional status as related to body composition and puberty. (K-4346-2)

PHOTO : Animal scientist Calvin Ferrell analyzes a blood derivative to estimate the uterine blood flow in pregnant cows. Blood flow may be a factor in fetal weight gain. (K-4326-2)

PHOTO : Animal geneticist Keith Gregory (left) and cattle operations manager Gordon Hays discuss care of heifers specially selected for their likelihood of producing twin calves. (K-4320-9)

PHOTO : Animal physiologist Robert Bellows observes body condition in different breeds on irrigated pasture. Body condition affects rebreeding performance. (K-4322-9)

PHOTO : Using an ultrasound scanner, animal physiologist Robert Staigmiller examines heifer's ovaries at puberty. (K-4322-20)
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Title Annotation:part 1
Author:Hays, Sandy Miller; Mazzola, Vince
Publication:Agricultural Research
Date:Nov 1, 1991
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