Symposium on Animal Genetics: XI11 International Congress of Genetics

MOLECULAR AND REPRODUCTIVE BIOLOGY IN ANIMAL GENETICS

R. B. CHURCH

Divisions of Medical Biochemistry and Biology, Faculty of Medicine, University of Calgary,

Calgary, Alberta, Canada

ROCEDURES in molecular and reproductive biology off er exciting new OS- Psibilities for the animal geneticist. In addition, many molecular biologists are now directing their expertise and techniques toward problems in animal genetics. Through the characterization of deleterious and normal gene expression an in- creased selection efficiency may be obtained along with an understanding of gene-environmental interactions and single gene contributions to phenotypic de- velopment. Examples of developments in these areas which have a place in animal genetics include:

a) The electrophoretic separation of proteins has had a definite influence on our approach to gene frequency studies in laboratory and wild animal populations (see HUBBY and LEWONTIN 1966).

b) Blood group analysis has been a very important tool in studies of gene drift in isolated populations of man (see MOTULSUKY 1970). Blood constituents may also be useful gene markers. For example, there is evidence that the B locus is associated with disease resistance and other economic traits in the chicken ( BRILES and ALLEN 1961 ) .

c) Somatic cell genetics, based on interspecies cell chimeras and the fusion of different cell lines within a single species, has permitted significant advances to be made in animal and human gene mapping in recent years (see EPHRUSSI 1972).

d) Chromosome banding techniques including the development of fluores- cence, Giemsa and DNA banding patterns in chromosome analysis has permitted positive identification of homologs (LIN et al., 1973).

e) Genome organization-the concept that the mammalian genome consists of a mixture of repeated and single copy DNA base sequences was introduced by BRITTEN and KOHNE (1968). This concept, based on the reassociation kinetics of denatured DNA and described by Cot, has been the basis for numerous evolution- ary studies (see MCCARTHY 1973).

f ) The transcriptional unit has been defined by RNA/DNA reassociation studies in estimations of the complexity of RNA transcription in differentiating tissues. The structure and processkg of the heterogeneous RNA synthesized at a transcriptional unit is discussed by PAUL and FIRTEL (1973; CHURCH and SCHULTZ 1973).

Examples of concurrent developments in reproductive biology which may have Genetics 78: 511-524 September, 1974.

512 R. B. CHURCH

a direct influence upon animal genetics include: a) Artificial insemination, a manipulation o€ the male gamete, has had the

greatest influence on animal breeding of any development in recent years. For example, progeny tests in numerous environments and with a constant genetic reference have changed the cattle industry. Possibly the greatest achievement ever made by the animal breeder has been the increase in milk production through the use of superior progeny tested sires in artificial insemination programs in the dairy herd (C. HENDERSON, personal communication, 1973).

b) Sex ratio control, or the separation of the X-bearing and Y-bearing sper- matozoon of an ejaculate has not been as rewarding as expected. It would appear that separation by differences in sedimentation rate, electrophoretic behavior or chromatographic techniques provide no better than a 60/40 sex ratio distortion at the present time (AUSTIN 1965).

C) Estrus cycle synchronization and better detection o€ estrus will be required for wider application of A.I. programs. In many species and in the bovine under suboptimal environmental conditions estrus control and heat detection are major obstacles to management (HENDRICKS and LAMOND 1972).

d) Preimplantation embryo manipulation, including the in uitro culture, fer- tilization, possible cloning, storage through freezing and implantation into foster mothers has recently been achieved in a number of mammalian species. These processes allow effective exploitation of the females’ genetic potential in animal breeding programs (ROWSON 1971 ) . Parthenogenesis permits the accumulation of a homogeneous genetic population which has desirable genetic potential. This has been achieved by OLSEN and Buss (1972) in turkeys and recently in mice by TARKOWSKI (1971). Homogeneous genetic stocks permit partition of the various components of variance in studies of gene-environmental interaction. Groups of full-sibs analyzed by any number of molecular techniques in any number of environmental conditions will provide better estimates of heritability and reestab- lish the importance of epistasis in genetic improvement.

I would like to discuss two areas of molecular and reproductive biology which we are studying in our laboratory; one involves the contribution which mammal- ian DNA base sequence heterogeneity may make to animal breeding and its pos- sible use in establishing a predictive “heterosis index” for the animal breeder; the second involves the use of preimplanation mammaliafi embryo manipulation to exploit maternal characteristics of reproduction in the bovine.

DNA BASE SEQUENCE HETEROGENEITY IN ANIMAL GENETICS

An animal cell possesses a DNA complement of sufficient size to specify up to several million different proteins. The reasons for this apparent excess of genetic material have eluded geneticists for many years. Recently, some insight into the complexity of the animal genome has been achieved through various reassociation experiments initiated by BRITTEN and KOHNE (1968). These workers suggest that multiple copies of some DNA sequences are interspersed between sequences present once per haploid genome in all animal cells. Some DNA sequences in

MOLECULAR A N D REPRODUCTIVE BIOLOGY 513

animal genomes were found to reassociate much more rapidly than others, and it was subsequently shown that these sequences were present in millions of copies per haploid genome. These results suggest that the genome contains many types of DNA sequences: those which are present in one or a few copies per genome referred to as a single copy or unique DNA sequence with structural functions; families of multiple copy DNA sequences collectively referred to as intermediate reiterated or repeated DNA sequences probably with a regulatory function; and a group of sequences present in a million or more copies which are not usually transcribed, consist of simple repeating units (see Walker 1973) and are usually present in the centromeric regions of the chromosome (see PARDUE 1973).

Reassociation kinetic characterization of the DNA base sequences present in a genome is expressed as Cot, a function of the nucleotide concentration and the incubation time (moles of nucleotide per litre per second) (BRITTEN 1970). Rep- resentative reassociation kinetic profiles of DNA of various species are shown in Figure 1. The highly repetitive DNA sequences reassociate at the lowest Cot

REASSOCIATION OF DNA FROM VARIOUS SOURCES

NUCLEOTIDE PAIRS

0

W U [L w 80 a

100 IO-^ 10-2 IO0 IO2 io4

Cot ( M x sec111

FIGURE 1.-The reassociation profiles of DNA isolated from various sources. Sheared, denatured mouse DNA at a concentration of 2 mg/ml was mixed with 3H-labelled single copy DNA sequences fractionated at Cot 220, at a concenwation of 1 fig/ml in conjunction with I4C E. coli DNA at a concentration of 10 ag/ml. The incubation reactions were carried out in 0.12 M sodium phosphate buffer, pH 6.8, at 60" for increasing incubation times. The percentage of DNA reassociated to double stranded structures was assayed by elution of the single stranded and duplex DNA by hydroxyapatite chromatography. The mouse, AT-rich satellite, was isolated from total mouse DNA by CsCl centrifugation and the preparation sheared to 460 nucleotide fragments before being denatured. The MS-2 and T, nucleic acid reassodation profiles were carried out after BRITTEN and KOHNE (1968). The exact details are outlined in CHURCH (1973).

514 R. B. CHURCH

values such that the very rapidly reassociating sequenccs present in the mouse genome reassociate prior to Cot 10-2-that is, about one million times faster than would be expected were these sequences present once per haploid genome. The intermediate reassociating DNA sequences, which seem to be transcribed at one time or another in development in one tissue or another (CHURCH and SCHULTZ 1974) and have a regulatory function in gene expression, reassociate through the intermediate range of Cot values from to I O 2 in mouse. The remaining 60% of the genome contains sequences which reassociate at a rate expected for sequences present once per haploid genome the size of mouse.

Ever since BRITTEN and KOHNE (1968) defined a “family of repeated se- quences” as those base sequences which are sufficiently related to reassociate with one another the relative importance of their structural localization in the genome and their functional correlation to quantitative genetic traits have interested quantitative geneticists. The size of the DNA base sequence family is described by the number of DNA base sequences which are sufficiently similar to reassociate at a given Cot value under specific incubation criteria (MCCARTHY and CHURCH 3 970).

Estimates of specific family size within genomes can be compared by consider- ing the rates of reassociation of DNA isolated from individuals of different inbred lines. The thermal stability of DNA/DNA duplexes, of either homologous or heterologous reactions, is similar, indicating similar base pair mismatching. The extent of hybridization reaction between DNA sequences isolated from different genetic lines provides, a way of comparing DNA base sequence family size in these genomes. Differences in base sequence family size in DNA isolated from individuals of different genetic lines is localized in the intermediate fraction of the chicken genome (SCHULTZ 1969; SCHULTZ and CHURCH 1973). Further evi- dence for the existence of DNA base sequence family “polymorphism” has been obtained from the reassociation kinetics of DNA isolated from individuals of inbred mouse lines (CHURCH, unpublished). Base sequence families which con- stitute a certain fraction of the genome in a particular individual may be present in greater or lesser amounts in the genome of another individual. Within the human genome, base sequence families detectable by in situ hybridization may be present in the DNA of one individual while being present in other individuals at a level which is not detectable by in situ hybridization (CHURCH unpublished). The quantitative differences between human satellite fractions seen in CsCl gradients may be reflections of differences in corresponding base sequence family sizes. These observations suggest the existence of DNA base sequence family polymorphisms in the genomes of individuals of any given species. This data is compatable with the idea that a conservative haploid DNA content is character- istic of individuals of a species, however many intermediate to fast sequence families are in a “dynamic equilibrium” within the same genome.

Several years ago, SCHULTZ (1969) undertook a study to determine the extent of intermediate base sequence family heterogeneity among individual full-sibs from inbred lines of chickens. The aim of this work was to identify inbred lines which were most divergent in DNA base sequence. Subsequently, on the basis of

MOLECULAR AND REPRODUCTIVE BIOLOGY

cot 0275 0 5 5 2 2 0 4 4 0 13 2

515

T

AA11

B B l l

cc11

FIGURE 2.-Heterogeneity in avian DNA sequences found in the intermediate DNA base sequence families isolated from individuals of 3 inbred lines. The reaction kinetics of DNA isolated f r m different inbred lines has been plotted to show the amount of reassociation occurring between each Cot value. The extent of reassociation orf AA1, BB11, and CC11 DNAs, respectively, is calculated in reference to the insolution reassociation of total chicken DNA isolated from the random bred control line designated T. The numbers in the blocks indicate the percentage of the total genome which reassociates to form a DNA/DNA duplex between the Cot values indicated. The exact conditions of the reassociation reactions have been reported by SCHULTZ ( 1969).

these, base sequence differences, he tested the relationship, if any, with the per- formance of biological crossbreds which had maximum productivity or heterosis. Our attempts to develop a predictive "heterosis index" based on DNA sequence families have been promising. The relative proportions of the genome contributed by various intermediate DNA base sequence families in different individuals of different inbred lines of chicken are shown in Figure 2. The rates of reassociation of DNA isolated €rom individual chicks representing genetic inbred lines desig- nated AA11, BB11, and CC11, can be distinguished. DNA isolated from AA11 individuals renatures more rapidly than does DNA isolated from the other lines at low Cot values and BBll and CCll DNA renatures to a greater extent than AA11 DNA at relatively higher Cot values. In similar experiments involving reciprocal crosses, we have found that a B1 male parent contributes more inter- mediate reiterated sequences than would be expected to the genome of a BCll individual than does the C1 female parent (SCHULTZ and CHURCH 1973). Al- though this does not necessarily mean that BCI l individuals will exhibit greater heterosis than CB11 individuals, it does suggest that there is an unequal genetic contribution of individual parents to the off spring as measured by reassociation analysis. A correlation between specific "strain crosses" which "nick" for quanti- tative traits, presumably due to epistasis, with DNA base sequence heterogeneity would be of interest. It should be stressed that in any hypothesis suggesting the existence of a dynamic state for intermediate reiterated DNA base sequence families, assurance of a constant quantity of DNA per haploid cell is required. Determinations of DNA content per nucleus in chicken cells have shown little variation (SOBER 1968). The observed variation in DNA base sequence hetero- geneity in the chicken genome may be associated with chromosomal fidelity since

516 R. B. CHURCH

some highly repeated sequences in mammalian species are associated with the centromeric regions of the chromosome (PARDUE 1973).

BRITTEN and DAVIDSON (1969) have suggested that gene duplication might produce “raw material” for the evolution of both regulatory and structural genes. These authors also claim that the repeated sequences are interspersed between single copy sequences within the transcriptional units of a genome (see PAUL 1973; DAVIDSON et al. 1973).

Although reassociation reactions involving intermediate DNA base sequence family components cannot be interpreted with as much certainty as would be desirable, they certainly indicate that DNA base sequence heterogeneity may have a very definite role in the regulation of gene action and a contribution to quantitative genetic characteristics. These initial attempts to establish a “heter- osis index” which would identify epistatic interactions between inbred lines in vitro before the biological crosses had to be made is promising. The potential abil- ity to assay the potential genetic contribution of any particular individual or line to the performance of hybrid offspring by in vitro techniques would increase effi- ciency of genetic improvement in animal selection programs.

THE ROLE O F GAMETE MANIPULATION IN A N I M A L GENETICS

Studies of gametogenesis and the preimplantation development in animal spe- cies have shown that preovulation oocytes can be recovered from ovaries, ferti- lized in vitro, cultured through a series of divisions and finally implanted into a synchronized foster mother for the rest of fetal development. The capability of freezing semen permitted extension of A.I. to general animal breeding programs. Recently the whole genome, the preimplantation embryo, has been successfully frozen, stored, thawed and implanted into a synchronized recipient mother. WILMUT and ROWSON (1973; using the basic embryo freezing technique de- veloped by WHITTINGHAM, LEIBO and MAZUR (1 972,1973) and WHITTINGHAM (1973) ; have frozen bovine embryos, thawed them and after implantation had a calf born from the foster mother. Manipulation of animal gametes increases the maximum genetic flexibility available to any animal breeding program, whether research or commercial. To utilize fully the genetic potential of superior females in any animal species is difficult, since very often the most productive females are not identified until they are past the end of their natural reproductive life- time. Most genetic studies of reproductive traits show low heritability estimates. In Table 1 are listed some of the experimental and fully developed manipulations of reproduction which may increase genetic flexibility in the male of an animal species. Manipulation of animal gametes permits greater genetic planning and, in conjunction with identification of genetic markers associated with high pro- duction, may have profound influence on the propagation of seed stock in animal improvement programs. Within the animal breeder’s own experimental herds or flocks, the possibility of having full-sib or “identical twin” populations of an- imals with which to study gene-environmental interactions has exciting possibil- ities.

MOLECULAR AND REPRODUCTIVE BIOLOGY

TABLE 1

Manipulation of reproductive processes in the male

517

Process Possibilities in animal eenetics

Artificial insemination (A.I.) Freezing and storage of semen

Progeny tests under many environmental

Control of sex ratio conditions

In vitro fertilization Control of estrus and heat detection

Now in practice in many species Widespread in bovine and man, not developed

Widespread in bovine, will be of equal

Little progress beyond 60/4Q ratio, of great

Limited to laboratory species at present Biggest obstacle to use of A.I. programs

in other species as yet

importance in other species

interest to cattle breeders

in many species

The separation of the X-bearing from the Y-bearing sperm in an ejaculate, with the aim of controlling the sex of the resulting off spring, has been an animal breeder's dream for many years. At present, the best results, whether by ultra- centrifugation, electrophoresis or chromatography, have been to shift the sex ratio to about 60/40. For economic feasibility such procedures should probably ensure greater than 90% sex certainty.

In contrast to the millions of sperm produced in the male each day only one ovum is produced per estrus in the cow. To utilize fully the thousands of oocytes which are present in each ovary of slaughter heifers, in vitro fertilization pro- cesses must be perfected. Spermatozoa. however, to be capable of fertilizing an oocyte, must go through a capacitation process in the female reproductive tract. Therefore before in vitro fertilization becomes a reality, conditions must be de- veloped which allow maturation of spermatozoa in vitro. Although in vitro fer- tilization has been achieved in laboratory species it is usually accomplished with spermatozoa recovered from the uterus. Therefore, for hr ther manipulation of the male gamete to be effective, some progress must be made in the manage- ment of estrus synchronization and knowledge of the reproductive tract in the female.

The manipulation of reproduction in order to achieve greater genetic flexibility in the female has not as yet been as successiul as it has in the male. Some of the reproductive processes which may be of importance to the animal breeder in the future are listed in Table 2. All of these procedures have been used in one species or another, therefore, their value in increasing the genetic flexibility available to the animal geneticist must be considered. To increase animal production and the efficiency of genetic improvement, better utilization of the female's reproduc- tive potential is required. For example, half of the world cattle population is used for milk production, and since the cow usually only carries one calf per gesta- tional period, nearly double the meat production could be possible if we were able to increase reproductive\ efficiency by multiple births either by genetic ma- nipulation, hormonal stimulation or through the non-surgical implantation of a recently thawed morula at the time of insemination. In the sheep industry, it is

518 R. B. CHURCH

TABLE 2

Manipulation of reproductive processes in the female

Process Possibilities in animal genetics

Estrus control and heat detection

Multiple births; through genetic, hormonal

Reliable supraovulation

Prepubertal oocyte recovery

o r implant procedures

Ova transfer or implantation In vitro fertilization, embryo culture,

sexing and storage

Blastomere and morulae fusion Parthenogenesis, cloning

Possible in bovine in near future, heat detection difficult on low plane of nutrition and health

Possible in bovine and ovine programs

Physiological barriers, many neural

Results not too promising, but potential

In use for limited gene pools in bovine, ovine Possible in laboratory species, required before

female genetic potential can be utilized like male through A.I.

factors to be defined

in reducing generation time

Possible in laboratory species Potential probably not limited to turkey and

mouse, of value to experimental populations

now common practice to utilize the reproductive proficiency of the Finnish Landrace to produce litters of off spring in crossbreeding programs.

Although multiple egg production from genetically superior females is pres- ently being used in embryo transplantation programs in the cattle industry, re- liable supraovulation aimed at inducing twins in beef and dairy herds has been in general a frustrating experience. The physiological barriers seem to be the net result of the health and nutritional status of the herd imposed upon the herd’s genetic background. The possibility of decreasing the generation time in a selec- tion program, through the recovery of prepubertal ova in species with a long generation time, exists although results have not been too promising to date. Suc- cess of blastomere or morulae fusion has been used successfully in the genetic analysis of development in the mouse ( GEARHART and MINTZ 1972).

Our laboratory has been involved in preimplantation embryo transfer in the bovine for several years in attempts to overcome the reproduction inefficiency of genetically desirable females. The heritability of reproductive characteristics is generally considered very low, hence, we initiated a search for genetic factors which affected reproductive performance. Genetically determined malforma- tions of spermatozoa are well known (DONALD and HANCOCK 1953), however, little research has been directed to such mutants in the female. GUSTAVSSON (1971) has reported that the frequencies of heterozygous and homozygous 1/29 chromosomal translocations are 13 and 0.5%, respectively, in Swedish Red and White cattle. The total reproductive implications of such chromosomal translo- cations are still somewhat obscure, although, GUSTAVSSON (1971) has reported an increased return to service in daughters of translocation-bearing bulls. Studies of repeat breeders in the “exotic” breeds have revealed four Robertsonian translo- cations; 1/29, 11-12/15-16, 2/4 and 11-12/17-18 (CHURCH and LIN, unpub-

MOLECULAR AND REPRODUCTIVE BIOLOGY 519

lished) . In one experiment, twenty-eight preimplantation embryos recovered from a 1/29 chromosomal translocation homozygote female were implanted into foster recipient mothers with complete failure to implant. When a further 24, two cell 1/29 homozygous translocation preimplantation embryos, were cultured abnormal cleavage occurred at the 3 4 blastomere cleavage stage.

Heifers which present as “repeat breeders” are usually suspected of hormonal imbalance. These females show regular estrus, appear to conceive as judged by anestrus for one or two cycles, only to return to estrus on a subsequent cycle. In the bovine with a long preimplantation period, 20 to 50 days, we believe that “repeat breeders” may often be the result of chromosomal aberrations or mutants which affect the embryo’s normal ability to develop and implant. Identification and selection against genetic aberrations which cause interruption of early em- bryonic development should improve the efficiency of animal production in those species which have long gestation periods and carry single off spring. However, some genetic traits, such as double muscling in bovine with its desirable growth rates, may be beneficial for the economic trait selected while being associated with reproductive inefficiency.

CONTRIBUTION O F EMBRYO IMPLANTS TO LIVESTOCK IMPROVEMENT

The possible contributions of embryo transplants or more correctly embryo implants to livestock improvement and animal genetics are listed in Table 3. Expansion of the limited gene pool of “exotic” beef cattle populations in Canada by embryo implantation is presently quite widespread. Limited quarantine sta- tion facilities for importation of breeding stock is responsible for the development of embryo implantation programs to expand gene pools. The proliferation of iden- tified genetic superiority in females by embryo implants offers the geneticist new opportunities in genetic flexibility in breeding programs. It is now possible to produce a number of full-sib sisters of the same age from cows who are near the

TABLE 3

Possible contributions of embryo implants to animal genetics

Possible contribution Location and species

Expand limited gene pools

Proliferate blood lines of genetically

Facilitate the transport of genetic potential superior females

Increase production by twinning Decrease the generation time in selection

To provide “A.I. technicians” by use of groups

Gene-environmental interaction studies;

programs by use of prepubertal females

of full-sib sires

pre- and postnatally

Anywhere demand greater than base

Progeny tested dairy and beef cow herds

Transpo’rt into N. America, New Zealand and Australia in rabbits or frozen

High level management, dairy cattle Species with long generation time

population

Range conditions for beef cattle, sheep

Laboratory animals, sheep and swine and cattle

520 R. B. CHURCH

end of their natural reproductive lifetime by the time data of their daughter’s performance can be evaluated by “female contemporary comparisons.” Beef pro- duction potential might be doubled by twinning all dairy cows through non-sur- gical implantation of frozen, thawed embryos at the time of insemination of the endogenous oocyte. Frozen embryos may be transported simply from one country or disease area to another as easily as semen is at the present time. The whole genome as opposed to the haploid male genome is involved which allows zygote- derived population development as a basis for foundation stocks in crossbreeding programs. Frozen embryo banks may be useful in establishing genetic pedigree standards and to act as controls for genetic drift in selection experiments. The frozen storage of valuable inbred lines of various animals, endangered species and mutant stocks would reduce cost of animal population maintenance. GEHR- ING (personal communication, 1973) has recently frozen Drosophila ovaries whose eggs subsequently produced fertile off spring after thawing and implanta- tion into larvae.

As a rancher, I am aware of the problems geography, nutrition and heat de- tection pose to the full use of the progeny-tested bulls available through A.I. Therefore, “four-legged A.I. technicians,” groups of full-sib bulls obtained through embryo implants from genetically superior donor cows, would be a wel- come addition to a breeding program. Four to five full-sib bulls, from a single implant operation, have been produced and will in the future be available to cattle breeders.

Embryo implantation, in the bovine, has several problems which hinder its use beyond valuable breeding stock at present. Some of the improvements in embryo manipulation which will enhance its use to animal geneticists and livestock breed- ers are listed in Table 4. Reliable and repeatable supraovulation is the most im- portant limitation to increased reproductive efficiency in the female. In an embryo implantation program, carried out in cooperation with Alberta Livestock Transplants, litters of up to 15 calves from a single donor operation have been carried through gestation by foster mothers. In our studies preimplantation em- bryos are implanted at the morula stage into synchronized recipient foster mothers, who then carry the exogenous embryo through gestation. The success of implantation of implanted embryos in the bovine averages about 60 percent.

TABLE 4

Improvements in embryo manipulations which will enhance genetic flexibility available to animal breeder

Total heat synchronization of donor and foster females Reliable and repeatable supraovulation Non-surgical recovery of embryos from donors and their non-surgical

The use of prepubertal virgin females or slaughterhouse supraovulated females In vitro fertilization Embryo freezing, storage and transport Embryo sexing, cloning, parthenogenesis

implantation into foster mothers

MOI,ECUI.AR AND REPRODUCTIVE BIOLOGY 521 TABLE 5

Possible eligibility requiremrnts of donor femnles to enhance maternal performance

Superior performance indcx in thc trait selected for No more than two breeding services for initial conception First three offspring born within 3 times gestation length Offsprings' contemporary comparsison above average No parturition tlificultics or reproductive irregularities No conformational nor tletectnble genetic defects

Optimal supraovulation: in our experience, is between 8 and 10 ovulations per supraovulated cycle in the donor female.

The prospect of recovering a higher percentage of the 3040,000 primary oocytes which are present in the ovaries of newborn females is a challenge to the reproductive physiologist. In an average lifetime a cow may carry less than 10 offspring, therefore, recovery of mature ova from supraovulated slaughter fe- males offers a fascinating prospect. The potential exploitation and utilization of this unused genetic potential is not presently available to the animal geneticist but is presently under study. Some of the possible eligibility requirements for females to qualify for genetic proliferation through embryo implantation pro- grams and thereby contribute to the reproductivc eficiency of the population are outlined in Table 5 . From this list of possible requirements it is evident that em- bryo implantation can be abused by animal breeders in propagation of reproduc- tive problems as well as be used for improvement of maternal characteristics.

Figure 3 illustrates 10 bovine morulae which were implanted into synchro- nized recipient females, which at the end of gestation resulted in a litter of 9

FIGURE 3.-Prciniplnritiition rmlwyos rccoverrd froin the rrprocluctive tract of a supra- owlated donor COW 5 clays aftrr fertilization. A total of 10 embryos were recovered and implanted in this operation.

522 R. B. C H U R C H

FIGURE 4.-A Limousin donor cow with her litter of 9 full-sib calves which are the result of implantation of 10 preimplantation embryos shown in Figure 3. The calves shown were carried through gestation and are now being m o t h e d by the Jersey and Holstein foster mothers which acted as recipients a t the time of embryo implantation.

calves. The genetic mother with the litter of full-sib offspring resulting from this one implant operation is shown in Figure 4.

I have included only a few of the molecular and reproductive biology advances which may be of value to the animal geneticist. It is now up to the animal geneti- cist to utilize these techniques to increase production efficiency through full uti- lization of the identified genetic potential of both males and females. The genetic flexibility and genetic planning which the animal breeder has within his grasp through these techniques is best illustrated by the cattle breeder who will be able to select a fully documented sire for use on groups of elite full-sib females pm- duced from his best performing cows by embryo implants to double selection response.

The assistance of Hyline Poultry Farms in supplying the inbred chicks and bovine embryo transplant experiments in cooperation with Alberta Livestock Transplants, Calgary, Alberta, is acknowledged as is the financial assistance of the National Research Council of Canada. Assistance of JUDY CROZIER, ANNE VIPOND and CAROL HOLLIDAY is acknowledged.

LITERATURE CITED

AUSTIN, C. R., 1965 Fertilization. Prentice Hall, Inc., Englewood Cliffs, N.J.

MOLECULAR A N D REPRODUCTIVE BIOLOGY 523

BRILES, W. E. and C. P. ALLEN, 1961 The B blood group system of chickens. 11. The effects of genotype on livability and egg production in seven commercial inbred lines. Genetics 46: 1273-1293.

BRITTEN, R. J., 1970 Repeated DNA and transcnption. pp. 187-216. In: Problems in Biology: RNA in Deuelopmmt. Edited by E. W. HANLEY. Univ. Utah Press, Salt Lake City.

BRITTEN, R. J. and E. H. DAVIDSON, 1969 Gene regulation for higher cells: a theory. Science 165: 319-357.

BRITTEN, R. J. and D. E. KOHNE, 1968 Repeated sequences in DNA. Science 161: 529-540. BRITTEN, R. J. and J. SMITH, 1970 A bovine genome. Carnegie Inst. of Wash. Yearbook. 68:

378-386. CHURCH, R. B., 1973 Methods for the study of hybridization and reassociation of nucleic acids

extracted from cells of higher animals. pp. 2W-301. In: Molecular Techniques and Ap- proaches in Deuelopmmtd Biology. Edited by M. CHRISPEELS. John Wiley and Sons, New York.

CHURCH, R. B. and G. A. SCHULTZ, 1973 Differential gene activity in the pre and post implan- tabon mammalian embryo. pp. 179-202. In: Current Topics in Deuelopmental Biology.

1974 Intermediate DNA base sequence heterogeneity. (In preparation.)

General interspersion of repetitive with non-repetitive sequence elements in the DNA of Xenopus. J. Mol. Biol. 77: 1-23.

DONALD, H. P. and J. L. HANCOCK, 1953 Evidence of gene-controlled sterility in bulls. J. Agr.

EPHRUSSI, B., 1972 Hybridization of somatic cells. University Press, Princeton, New Jersey. GEARHART, J. D. and B. MINTZ, 1972 Clonal origins of somites and their muscle derivatives:

evidence from allophenic mice. Devel. Biol. 29: 27-37. GUSTAVSSON, I., 1971 Distribution of the 1/29 translocation in the A.I. bull population of

Swedish Red and White cattle. Hereditas 69: 101-106. HENRICKS, D. M. and D. R. LAMOND, 1972 Hormonal interrelations in beef cows with induced

multiple ovulations. Nature 235: 2%23. HUBBY, J. L. and R. C. LEWONTIN, 1966 A molecular approach to the study of genic heterozy-

gosity in natural populations. I. Number of alleles at different loci in Drosophila subobscura. Genetics 54: 577-594.

Karyology of cells in culture. pp. 773-787. In: Tissue Culture, Methods and Applicafions. Edited by P. F. KRUSE and M. K. PATTERSON. Academic Press, New York.

Vol. 8. Edited by A. MONROY and A. MOSCONA. Academic Press, New York. - ,

DAVIDSON, E. H., B. R. HOUGH, C. S. AMENSON and R. J. BRITTEN, 1973

Sci. 43: 178-181.

LIN, C. C., I. A. UCHIDA, F. A. ARRIGHI, H. C. WONG and S. FEDOROFF, 1973

MCCARTHY, BRIAN, 1974 Evolution of DNA sequences. Genetics 78: 000-000.

MCCARTHY, B. J. and R. B. CHURCH, 1970

MOTULSKY, A. G., 1970

Specificity of hybridization. Ann. Rev. Biochem. 39:

Biochemical genetics of hemoglobins and enzymes as a model for birth defects research. pp. 199-208. In: Congenital Malformations. Edited by F. C. FRASER and V. A. MCKUSICK. Excerptia Medica, New York.

Segregation of two alleles for color of down in partheno- genetic and normal turkey embryos and poults. Genetics 72 : 69-75.

131-150.

OLSEN, M. W. and E G. Buss, 1972

PAUL, JOHN, 1974 PARDUE, M. L., 1974 ROWSON, L. E. A., 1971

26: 113-126.

The transcriptional unit in Eukaryotes. Genetics 78: (In press.) Repeated sequences in eukaryotic chromosomes. Genetics 78: (in press.)

The role of reproductive research in animal research. J. Reprod. Fert.

524 R. B. CHURCH

SCHULTZ, G. A., 1969 Base sequence heterogeneity in avian DNA. Ph.D. thesis, University of Calgary.

SCHULTZ, G. A. and R. B. CHURCH, 1974 Transcriptional patterns in early mammalian develop- ment. In: The Biochemistry of Animal Development. Vol. 3. Edited by R. WEBER. Academic Press, New York. (In press.)

SOBER, H. A., 1968 Handbook of Biochemistry, Selected Data for Molecular Biology. p. 257. Chemical Rubber Co., Cleveland, Ohio.

TARKOWSKI, A. K., 1971 Parthenogenesis in the mouse. J. Reprod. Fert. (Suppl.) 14: 31-39. WALKER, P. M. B., 1973 Multiple genes and repetitive DNA. Genetics 78: OCKk0130.

WHITTINGHAM, D. G., 1974

WHITTINGHAM, D. G., S. P. LEIW and P. MAZUR, 1972

WILMUT, I. and L. E. A. ROWSON, 1973

Embryo banks in the future of developmental genetics. Genetics

Survival of mouse embryos frozen to

Experiments on the low temperature preservation of

78: 3 9 5 4 2 .

-196" and269". Science 178: 411-414.

cow embryos. Vet. Record 92 : 68e690.