for natalia, pablo and cintia

FOR NATALIA, PABLO AND CINTIA

DECLARATION

I declare that this thesis was composed by me and is a report of my

analyses of data which were collected by members of the staff of the ARC

Animal Breeding Research Organisation.

Andres E. Carden

University of Edinburgh

November, 1982

ABSTRACT

This thesis investigates different genetic aspects of susceptibility to halothane anaesthesia in pigs. The mode of Inheritance of the reaction to halothane was investigated in a Pietrain/Hampshire (PTH) and a British Landrace experimental herds. The single-recessive mode of inheritance was tested as a hypothesis in the context of (I) a single-locus-two-alleles model where both the heterozygote and one homozygote react to the anaesthetic and (II) a two-locus model involving a susceptibility locus and a suppressor locus. Maximum likelihood techniques were used to fit the models to the data. The results of the single-locus analysis did not disprove the single-recessive hypothesis in PTh. The same analysis rejected a strictly recessive mode of inheritance In Landrace. The two-locus analysis in PTR indicated that the addition of a suppressor locus to a single recessive model could Improve the explanation of halothane testing results. The two-locus analysis also rejected the single-recessive hypothesis in Landrace. A study was conducted to estimate heritability of the time of onset of reaction to halothane. Estimated heritabilities at eight weeks of age were 0.65 ± 0.59 in PTH and 0.12 ± 0.58 in Landrace, after half-sib analyses. The precision of these estimates was too low to allow firm conclusions to be drawn. There were clear indications that a 3-minute halothane test may be too short to detect all positive reactors in the British Landrace breed.

The effect of age on incidence of susceptibility was studied on pigs which were repeatedly exposed to the anaesthetic. In three trials, PTH and Landrace pigs were given three 3-minute halothane tests, at 19, 35 and 54 days of age. In a fourth trial Landrace pigs received four 5-mm tests, at 21, 35, 49 and 63 days. All pigs were offspring of positive x positive matings. Maximum likelihood estimates of the probability of positive reactions were 0.47, 0.73 and 0.88 at the three ages in PTH; 0.20, 0.63 and 0.60 in the first Landrace trial and 0.64, 0.86, 0.84 and 0.90 in the second Landrace trial. Thus, In all trials penetrance increased with age. In both breeds, and at all ages, penetrance was lower In males than in females.

Two studies are presented, assessing effects of susceptibility on reproductive, growth and body composition traits. They evaluate differences between reactor and non-reactor PTH pigs from two lines selected for and against susceptibility: SS and SR respectively. The first study analyses litter records from females of the SS and SR lines. At weaning susceptible females produced about 1.5 piglet/litter less than normal females. This was apparently due to both smaller litters at birth and higher piglet mortality during lactation. The second study analyses results from three trials in which putative heterozygotes at the halothane locus were compared with putative normal homozygotes. There. were no significant differences in growth traits. Heterozygotes had paler meat. There were indications that heterozygotes have about 1.5 % more lean tissue in the carcass. The small sizes of the SS and SR lines proved to be an important source of error in the estimation of differences associated with halothane susceptibility.

CONTENTS

CHAPTER 1

GENERAL INTRODUCTION I

CHAPTER 2

THE GENETICS OF HALOTHANE SUSCEPTIBILITY IN PIGS:

A REVIEW OF THE LITERATURE

I. Malignant hyperthertuia 3

II. The biochemical bases of the malignant

hyperthermia reaction 5

III. The halothane test 6

IV. Genetics of halothane susceptibility

Mode of inheritance 7

Linkage relationships 9

V. Associated effects on production traits

Comparison of phenotypes 14

Comparison of genotypes 16

VI. Incidence of halothane susceptibility 19

VII. Practical Implications 21

VIII. Questions for future research 22

Page

Page

CHAPTER 3

THE INHERITANCE OF HALOTHANE SUSCEPTIBILITY

IN PIGS

Introduction 24

Material and Methods

Animals 25

Models

Model 1. Single-Locus 27

Model 2. Two-Locus 29

Computations 32

Results

Model 1. Single-Locus 33

Model 2. Two-Locus 37

Discussion 37

CHAPTER 4

STUDIES ON THE TIME OF ONSET OF REACTION

TO HALOTHANE ANAESTHESIA

Introduction 48


Animals 48

s-s4of4.1 ia Lyes

Analysis of heritability 50

Analysis of repeatability 50

Results

Analysis of heritability 54

Analysis of repeatability 57

Discussion 60

Page

CHAPTER 5

THE EFFECT OF AGE ON HALOTHANE SUSCEPTIBILITY

Introduction 68


Experimental 68

Statistical analysis 70

Results

Effects of previous tests on subsequent ones 77

HP reactions 78

RD reactions 78

HP + RD reactions 81

Discussion 81

CHAPTER 6

THE EFFECTS OF HALOTHANE SUSCEPTIBILITY ON SOME

ECONOMICALLY IMPORTANT TRAITS 87

6a THE GENETIC STRUCTURE OF THE ABRO—PTH LINES

Demographic structure of the PTH population 88

The genetic composition of the SS and SR lines 89

Random genetic differentiation of the lines 95

b DIFFERENCES IN REPRODUCTIVE TRAITS BETWEEN HALOTHANE

SUSCEPTIBLE AND HALOTHANE TOLERANT PIGS 100


Animals 101

Statistical analyses

I. Analysis of litter size and piglet weight 102

Within—line matings: all records 103

Within—line matings: first parities 103

Carrier trials 103

Variance of line differences 104

II. Analysis of piglet mortality 106

Results 107

Discussion 107

6c DIFFERENCES BETWEEN PIGS OF PREDICTED GENOTYPES AT

THE HALOTHANE LOCUS IN GROWTH AND CARCASS TRAITS 113


Animals 114

Performance testing 115

growth traits 116

Carcass traits 116

Ruinpback traits 117

Full dissection traits 119

Statistical analyses

Analysis of growth, carcass and rumpback

traits 119

Analysis of the full dissection results 121

Variance of genotypic differences 124

Results 125

Discussion 127

Page

CHAPTER 7

CONCLUDING REMARKS 140

The mode of inheritance of susceptibility 140

Changes in productivity traits associated

with susceptibility 145

APPENDIX

149

ACKNOWLEDGEMENTS

REFERENCES

CHAPTER 1. GENERAL INTRODUCTION

The discovery of a gene with detectable effects on metric characters

in pigs has prompted a considerable amount of research in recent years.

The existance of a genetic locus with major effects on muscularity was

notably foreseen by 011ivier (1968) after studies with Pietrain pigs.

More recently, research with blood-type markers indicated a major gene

affecting meat quality traits (Jensen et al., 1976). The main

breakthrough, however, was the finding that the malignant hyperthermia

reaction, as triggered by the anaesthetic halothane, was largely

controlled by a single autosomal locus (011ivier, Sellier and Monin,

1975). It has been known for some years that pigs differing in their

susceptibility to halothane anaesthesia differed in meat quality traits

and in their tolerance to stress (Harrison, 1972); it was soon shown that

they also differed in lean content (Eikelenboom, Minkema and van Eldik,

1976). Thus, the discovery of the halothane gene not only confirmed the

existence of a locus with major effects on production traits but also

provided pig breeders with a simple screening tool. A review of the

literature on halothane susceptibility in pigs is presented in Chapter 2.

A research project was started in 1974 at the Animal Breeding Research

Organisation (ABRO), Edinburgh, to investigate several aspects of the

halothane susceptibility phenomenon in pigs. Over the years the project

has generated a comprehensive body of information part of which -a

combination of experimental results and field records' collected

routinely at ABRO constitutes the material for the studies in this

thesis. The investigations presented here can be divided into two groups.

The first group comprises studies dealing with the reaction to halothane

anaesthesia: the mode of inheritance of this reaction (Chapter 3), the

genetics of time of onset of reaction (Chapter 4) and the effects of age

on halothane susceptibility (Chapter 5). The second group (Chapter 6)

comprises studies dealing with the effects of halothane susceptibility on

reproductive, growth and body composition traits.

CHAPTER 2. THE GENETICS OF HALOTHANE SUSCEPTIBILITY IN PIGS

A REVIEW OF THE LITERATURE

This review deals with the problem of halothane susceptibility in pigs

focussing mainly on the genetic aspects of this trait and its connection

with different production characters. The literature on malignant

hyperthermia was extensively reviewed by Gronert (1980). Particular

aspects of the problem in pigs were also reviewed: the mode of

inheritance was discussed by 011ivier (1980); the practical applications

of the halothane test were reviewed by Webb (1981); Smith (1981 a)

considered practical breeding aspects.

I. MALIGNANT HYPERTHERIEIA.

Harrison et al. (1969) and Sybesma and Eikelenboom (1969) were the

first to report that some pigs, after exposure to halothane anaesthesia,

undergo a characteristic reaction called malignant hyperthermia or

malignant hyperpyrexia. The main clinical symptoms of the malignant

hyperthermia (MM) reaction are:.

1— Dramatic rise in general metabolism, leading to increases in body

temperature and acidosis.

Enhanced muscle cell permeability, resulting in increased potassium,

ionized calcium, sodium, creatine-phosphokinase and myoglobin

serum concentrations.

Sympathetic stress response.

Gross muscle contracture.

Tachicardia and decreased heart output.

The symptoms have been described in great detail by Gronert (1980);

the reaction is lethal if the administration of anaesthetic is not

suspended in the early stages; the animal dies from cardiac failure.

In spite of some differences, the MH syndrome in the pig is very

similar to the malignant hyperpyrexia syndrome in humans (Wingard and

Gatz, 1978; Gronert, 1980); in fact, the pig provides a useful model for

the study of the human syndrome. In addition, similar reactions have been

reported in the dog, in the cat and in the horse (Short, 1978). In pigs,

susceptibility to halothane appears to be a manifestation of a more

general disorder known as Porcine Stress Syndrome or PSS (Harrison, 1972).

Any strain to which susceptible pigs are exposed can lead, through the MH

reaction, to sudden death. Thus excsrcse, excitement, high ambient

temperature, anoxia can all trigger MH episodes (Briskey, 1964; Sybesma

and Eikelenboom, 1969; Schulman, 1981). Drugs like succinyleholine,

different volatile anaesthetics and some sympathetic agonists are also

triggering agents of varying effectivity (Gronert, 1980).

II. THE BIOCHEMICAL BASES OF THE MALIGNANT HYPERTHERNIA REACTION.

It will be useful to review briefly the biology underlying the MH

syndrome. Although there is controversy about the specific metabolic

error in stress susceptible pigs it is nowadays widely accepted that the

defect lies in the skeletal muscle and that there may be a generalized

alteration in membrane properties affecting calcium movements, perhaps

involving specific enzyme or cell membrane structural protein variants

(Gronert, 1980).

Different studies have shown that there is an enhanced Ca++ efflux

from mitochondria (Cheah. and Cheah, 1976; 1978) and an impaired Ca-f-I-

transport in the sarcoplasmic reticulum of stress susceptible pigs

(Campion and Topel, 1975; McIntosh, Berman and Kench, 1977). Other

studies have shown increased erythrocyte fragility in susceptible pigs

(Harrison and Verburg, 1973; King , 011ivier and Basrur, 1976) which

suggests there are differences in membrane osmotic properties. This type

of evidence supports the hypothesis of a generalized membrane disorder

affecting Ca++ movements.

Briefly, the hypothesis states that after certain stimuli, for

example muscle fibre depolarization by some drugs or increased muscular

activity ; there follows, in susceptible pigs, an uncontrolled rise in

intracellular Ca-H-, the ultimate cause of which is still unknown. This

would in turn cause a rise in general metabolism -an homeostatic attempt

to lower the high intracellular Ca-H- concentration. This situation would

trigger the sympathetic stress response. The process would finally

precipitate into the known series of events: respiratory and metabolic

acidosis, increased heat production, decreased heat loss, further loss of

Ca++ control resulting in muscle contracture, generalized failure of cell

membranes and cardiovascular collapse. The theory awaits confirmation of

the increased intracellular Ca++ levels. For a detailed description of

the theory and a discussion of alternative hypotheses, see the review on

malignant hyperthermia by Gronert (1980).

III. THE HALOTHANE TEST.

The use of halothane anaesthesia to identify pigs which are liable to

PSS is known as the 'halothane test. It consists in administering the

animals a mixture of oxygen and halothane through a facial mask. Pigs

developing a clear rigidity of the hind limbs are classified as halothane

positive or stress susceptible. As soon as this symptom is observed the

administration of anaesthesic is interrupted. Most positive pigs react

within the first three minutes; the mean reaction time is about 100

seconds (Bulla et al., 1979; Webb and Jordan, 1979; Fr ,p'ystein at al.,

1981). Mortality during testing varies from around zero to 12 % of

positive reactors, depending on breed and environmental factors (Webb,

1981).

In experiments the flow of oxygen, the concentration of halochane and

the duration of the test have been somewhat variable. In general terms

however the oxygen flow was of the order of 3 1/mm, with a halothane

concentration of about 5 %. The test is usually extended for a period of

3 to 5 minutes (Eikelenboom, Minkema and Sybesma, 1978). Those pigs

remaining relaxed throughout are classified as halothane negative or

stress resistant.

Alternative methods for predicting liability to PSS, like the

measurement of plasma creatine-phosphokinase (CPK) activity, the study of

genetic markers in blood or the erythrocyte fragility test were discussed

by Allen et al. (1980) and by McGloughlin (1980); see also the

Proceedings of a recent symposium (FrØystein, Slinde and Standal, 1981).

IV. GENETICS OF HALOTHANE SUSCEPTIBILITY.

(i) Mode of inheritance.

The hereditary aspects of porcine susceptibility to halothane were

recognized early on by Allen et al. (1970); Christian (1972) and Hall,

Trim and Woolf (1972). When the trait is simply defined as 'hind limbs

rigidity after halothane application the phenotypic variation is

discontinuous. Apart from a few doubtful cases there are two classes:

reactors and non-reactors. Christian (1972) suggested that this reaction

was inherited as a single autosomal recessive gene with incomplete

penetrance. Subsequently 011ivier, Sellier and Monin (1975); Minkema,

Eikelenboom and van Eldik (1976); Smith and Bampton (1977); McPhee,

Takken and DArcy (1979); Lnccher ; Schneider and Jucker (1979) and

Schepers (quoted by Simon, 1980) all fitted single autosomal recesive

models to data from planned matings or from field observations. The

conclusions from these studies agree in that halothane susceptibility is

likely to be determined by the recessive homozygote genotype at a single

locus, this genotype having incomplete penetrance (0.9 as averaged by

Webb, 1981). It must be pointed out that, apart from McPhee et al.

(1979) who compared recessive and dominant single gene hypotheses, the

studies above did not produce statistical evidence for rejecting

alternative models of inheritance.

Simon (1980) argued that it would be difficult to differentiate

between the single recesive hypothesis and a quantitative-threshold model.

However, the evidence that halothane sensitivity is correlated with

several blood markers belonging to a single linkage group (Andresen and

Jensen; 1977; JØrgensen, 1981) suggests that there is a locus with major

effects on this trait. Simple genetic mechanisms therefore are most

likely but, so far, only the single recessive model has been properly

explored. The possibility that penetrance is under genetic control -as

suggested by the pattern of response to selection in favour of positive

reactions in Pietrain/Hampshire pigs at ABRO (Webb, 1981)- cannot be ruled

out.

When halothane susceptibility was defined in a way different from

above, or when the test procedures were varied, a different mode of

inheritance was also inferred. Hall et al. (1972) treated the pigs with

a combination of halothane and succinyicholine. Their tentative

conclusion was that the NH reaction, after such a test, is controlled by a

single autosotnal dominant gene. Jones et al. (1972) identified halothane

susceptible animals by measuring muscle ATP depletion during exposure to

the anaesthetic in vitro; from the incidence observed within a familiy

they inferred a single autosomal dominant pattern of inheritance. Williams

et al. (1975; 1978) considered rapid heart beat, muscle rigor and/or

rise in rectal temperature' to be the signs of positive reactions. They

proposed a 'strongly modified single dominant gene or two dominant genes

acting in concert as the hereditary mechanism for halothane

susceptibility. Britt, Kallow and Endrenyl (1975) recognized five

different classes of response in skeletal muscle biopsies treated with

caffeine or caffeine plus halothane. They concluded that this trait might

be controlled by two loci but did not specify any particular genetic model

nor presented a clear genetic analysis of their observations. In general

terms the latter reports are characterised by a vague definition of the

trait under study and by a less rigorous testing of genetic hypothesis or

no hypothesis testing at all.

For practical agricultural purposes it seems fairly reasonable to

define the trait as hind limbs rigidity after halothane application and

to assume a single-recessive mode of inheritance, at least until clear

evidence is presented on the contrary. However, the trait could be

defined at a higher level of resolution; the possibility that in such

instances it might have a different mode of inheritance cannot be ruled

out.

There is an effect of age on the expression of susceptibility to

halothane. After repeated tests Webb (1980 a; 1981) has shown that the

penetrance of the susceptible genotypes increases with the age of the

pigs. This effect could complicate the interpretation of studies on mode

of inheritance, if the pigs were young or varying in age. It also could

account for the variable penetrance estimates reported in the literature.

(ii) Linkage relationships.

Andresen (1971) was the first to report a linkage group involving

three loci in pigs. These were the locus for a red blood-cell antigen, H,

and the loci controlling two enzymatic polymorphisms: phospho-hexose

isomerase (PHI) and 6-phosphogluconate-dehydrogenase (6-PGD).

Later it was discovered that the locus for a gene having major effects

on halothane susceptibility belongs to the same group (Rasmusen and

Christian, 1976; Andresen and Jensen, 1977). Andresen and Jensen (1977)

called this locus HAL. According to Rasmusen, Beece and Christian (1980)

a fifth locus -an inhibitor of the A-0 system- might also belong to the

same linkage group.

Seven alleles have been recognized within the H system (Rasmusen,

1975); for each of the enzyme loci two codominant alleles are known

(Gahne, 1979). HAL comprizes two alleles, designated N and n by Minkema

et al. (1976). Details of the loci are summarised in Table 2.1. Table

2.2 shows the recombination frequencies found in a recent study involving

several Landrace strains (Jrgensen, 1981). From the values in Table 2.2

the most likely sequence for the four loci in the chromosome is:

PHI - HAL - H - 6-PGD

Several population studies have found linkage disequilibria amongst

1

the components of the PHI-HAL-H system. In these investigations HAL has

always been associated with PHI B

(Guerin, 011ivier and Sellier, 1978;

Watanabe et al., 1978; Andresen, 1979; Andresen et al. ) 1980;

Jyrgensen, 1981) even though five different breeds where involved. The

A gamete PHI - HAL has not yet been detected in Danish Landrace

(JØrgensen, 1981). Similarly, linkage disequilibria were also reported

Table 2.1. Alleles at the different loci in the 5-loci linkage group.

System Alleles

H : a, b, ab, cd, bd, be, and -;

or simply a and -

PHI: A and B

6-PGD : A and B

HAL: N and n

A-O suppresor : S and s

Table 2.2. Per cent recombination frequencies (8)

among the polymorphic systems HAL, PHI,

H and 6-PGD in Landrace pigs (taken from

Jrgensen, 1981).

System 8 S.E.

HAL - PHI 0.0

HAL - H 3.0 (2.3)

HAL - 6-PGD 9.1 (6.3)

PHI - H 4.5 (1.7)

PHI - 6-PGD 12.4 (4.6)

H - 6-PGD 5.2 (1.4)

between the HAL and H loci (Andresen et al., 1981; Frystein et al, 1981;

Jrgensen, 1981). The results from these studies, as well as evidence

from other investigations (Hjny et al., 1979; Imlah and Thompson, 1979;

Schulman, 1981) indicate that HAL is generally associated with the H a

allele.

Depending on the existence of linkage disequilibria the PHI and H

systems could be used, as markers of the HAL locus, to identify stress

susceptible pigs and to reduce the frequency of HAL " by selection.

Jrgensen (1981) calculated, that selection against those pigs carrying the

Ha allele and with the PHI -PHI genotype could have the saiue.result as

culling of the halothane reactors in the Danish Landrace breed, but in

order to achieve this the number of pigs to be removed from the population

should be considerable higher in the former case.

The correlations found between H or PHI types and several traits

-mainly carcass traits - are now attributed to the disequilibria in

different populations among these loci and HAL, which is believed to be

the locus directly responsible for the differences (JØrgensen and

Hyldgaard-Jensen , 1981). The maintenance of such disequilibria in pig

populations has been attributed to selection favouring the heterozygote at

the HAL locus, disequilibria already present when selection started and a

hitch-hiking effect (Guerin. 011ivier and Sellier, 1979; 011ivier, 1980).

It remains to be explained why, when disequilibria exist, they have always

the same sign, across so many different breeds. Guerin et al. (1979)

speculated that this could indicate that the HAL 1 genes in those

populations all derive from a common origin.

V. ASSOCIATED EFFECTS ON PRODUCTION TRAITS.

A considerable volume of research indicates that the HAL locus has

effects on a variety of traits, many of which are economically important.

These studies can be divided into two types: comparison of phenotypes and

comparison of genotypes.

(i) Comparison of phenotypes.

This was the most common type of investigation. Reactor and

non-reactor pigs were compared for a variety of traits. The results of

such comparisons will depend on the genotypic values and frequencies, they

are therefore likely to vary across populations.

Some results from the literature are summarised in Table 2.3. The

reported differences were pooled after weighting them according to numbers

and variances. The statistical significance of the pooled differences was

determined by means of a t-test. Such a table may undoubtedly remove

important differences in particular breeds or feeding regimes, and the

pooling of data from heterogeneous populations can be misleading. It

could be useful, though, as a general summary of the available

information.

While there were no clear effects in growth rate, food conversion or

appetite, the differences in body composition and meat quality traits were

important and consistent. Halothane susceptible pigs were leaner,

slightly shorter and had a higher carcass yield. Although much variation

in meat quality traits depends on pre-slaughter conditions (Malmfors,

Table 2.3 Some differences in performance between halothane positive (HP)

and halothane negative (HN) pigs, summarised from the literature.

No. No. Difference Trait •studies pigs HP-HN (S.E.) Author #

Growth

Growth rate (g/day) 9 4937 -0.1 ( 3.21) 1,2,3,5,7,8,9,13,14

Food conversion (food/gain) 5 3256 0.0 ( 0.03) 1,2,8,9,14

Food intake (g/day) 2 2922 -8.0 (17.21) 1,2

Body composition

Carcass yield (%) 5 1682 0.7 (0.32)* 1,2,7,12,14 Lean content (%) 9 1289 3.2 (0.50)** 1,3,5,6,9,11,12,13,14 Carcass length (mm) 6 1710 -9.0 (3.21)** 1,2,6,7,12,14

Meat quality

Muscle pH (45 mm) 7 1027 -0.3 (0.04)** 1,3,6,9,10,12,13 Muscle pH (>24 h) 5 371 0.0 (0.04) 1,4,6,7,10 Gofo index 4 943 -8.4 (1.24)** 3,9,10,13 Transmission value 4 1502 16.2 (2.65)** 1,2,6,12

* (P<0.05) ** (P<0.01)

# References: (1) Carlson et al.(1980); (2) Eikelenboom et al.(1978); (3) Gerwig, Vögeli and Schwörer (1979);(4)Jensen and Andresen (1980); (5) Kovach (1980); (6) Kukoyi et al.(1981); (7) Monin, 011ivier and Sellier (1976); (8) 011ivier, Sellier and Monin (1978); (9) Rogdakis, Ensinger and Faver (1980); (10) Schmidt and Kaliweit (1979); (11) Schulman (1981); (12) Verstegen et al. (1976); (13) Vdgeli (1978); (14) Webb and Jordan (1978).

1981) the defect in pork quality known as Pale, Soft, Exudative (PSE) is

to some extent associated with halothane susceptibility. In fact, the

halothane test was originally introduced as a predictor in vivo of PSE

muscle (Eikelenboom and Minkema, 1974). Webb (1981) reported that the

difference in incidence of PSE between reactor and non-reactor carcases

from the literature averaged 46 %. Table 2.3 shows that muscle from

reactor pigs has a faster post-mortem decline in pH, paler colour as

indicated by the Göfo index and higher protein solubility which have been

used as objective indicators of PSE (Barton-Gade, 1981).

In addition to these results other studies indicated that positive

reactors had a different visual conformation (011ivier et al., 1978) and a

higher post weaning mortality (Eikelenboom et al., 1978; Webb and Jordan,

1978). Furthermore, halothane susceptible sows would produce smaller

litters (Webb and Jordan, 1978). Webb (1981) estimated that the higher

mortality and reduced litter size would offset, under British conditions,

the commercial advantages of higher carcass yield and lean content.

(ii) Comparison of genotypes.

These are based, of course, on the single. recessive model of

inheritance. There were fewer studies of this type; in some of them the

pIgs were classified into genotypes after so many assumptions that their

conclusions should be taken cautiously. Some of the results in the

literature are summarised in Table 2.4 from Webb (1981), giving equal

weight to each study.

As far as growth rate and food conversion are concerned, the results

Table 2.4. Some differences in performance between halothane genotypes,

summarised from the literature (Webb, 1981).

Difference

nn-NN Nn-NN No.

Trait studies X range X range Author Ii

Growth rate (g/day) 5 -11 -102 to 42 3 -69 to 47 2,3,5,6,7

Food conversion 2 0.00 -0.01 to 0.01 0.02 0.01 to 0.03 2,7

Carcass yield (%) 2 1.3 0.9 to 1.7 0.8 0.1 to 1.4 2 11 7

Lean content (%) 5 3.6 2.7 to 4.3 1.8 1.0 to 2.3 1 , 3 3, 4 9 6 31 7

Carcass length (mm) 6 -10 -1 to -17 1 -8 to 12 1 31 2,3,4,6,7

P.S.E. (Z) 3 51.8 49.6 to 51.9 1.6 0.4 to 3.2 1,3,7

I! References: (1) Andresen, Jensen and Barton-Gade (1981); (2) Eikelenboom

et al. (1980); (3) Jensen (1981);(4) Jensen and Andresen (1980);(5) Liescher,

Schneider and Jucker (1979);(6) Schneider, Schwrer and Blum (1980); (7) Webb

(1981).

in the literature appear inconclusive. There seems to be some indication,

though, that the heterozygote may be intermediate for lean content and

carcass yield. The effects of the gene on carcass length and meat

quality, on the other hand, appear to be recessive, as well as those on

litter size as reported by Schneider et al. (1980). Based on the data of

Eikelenboom et al. (1980) Brascamp, Eikelenboom and Minkema (1980)

estimated that the halothane locus would account for some 60 and 20 Z of

the additive genetic variance in meat quality traits and in lean content

respectively, in the Dutch Landrace breed. According to Webb (1981) the

heterozygote would offer economic advantages when compared with both

homozygote genotypes under British conditions. The locus would show

overdominance in those environments where leanness is an economic goal

(Andresen et al., 1981).

It seems clear that the halothane locus is marking a region in the

chromosome with major effects on traits related to the amount and

physico-chemical properties of muscle tissue, fitness and prolificacy.

However, the genetic nature of the relationship between halothane

susceptibility and the changes in those traits is not clearly established

yet. Are all the differences a cascade of effects arising from a unique

physiological defect -are they pleiotropic effects of the same gene ? or

are some of them the result of linkage disequilibria among the halothane

locus and other loci ? After theoretical considerations the hypothesis of

pleiotropy is generally favoured (Guerin et al., 1979; 011ivier, 1980;

Jrgensen and Hyldgaard-Jensen, 1981) although there is not much

supporting evidence so far. On the other hand the finding that the H

locus accounted for about 6 % of the variance in reproductive traits in

Duroc Jersey pigs (Jensen et al., 1968) raises some doubts. It was found

in that study that the Ha allele produced a reduction in litter size;

the same effect was also attributed to HAL ' (Webb and Jordan, 1979;

Schneider et al., 1980). As it was pointed out before, both genes are

generally found positively associated. The important point is that the

present frequency of HAL seems to be negligible in the Duroc Jersey

breed.

The resolution of the dilemma as to whether the effects on performance

arise through plelotropy or linkage will require finer analyses. However,

a proper answer to this question is desirable even from a practical point

of view. Perhaps it would not be impossible to dissociate some of the

beneficial and harmful effects by means of selection of the appropriate

recombinants.

VI. INCIDENCE OF HALOTHANE SUSCEPTIBILITY.

There seems to be a wide range of incidence of reaction to halothane

in the different worlds pig populations. Apparently Large White and the

American breeds would have very low incidences. Most Landrace strains

show intermediate frequencies while Pietrain and Belgian Landrace appear

to have high incidences. Webb (1980 b; 1981) has presented a table

summarising the incidence in different worlds breeds as reported in the

literature. Franceschi and 011ivier (1981) presented a similar table.

Table - 2.5 - is a summary taken from Webbs table. Some of the results are

from field surveys, others from testing stations or from experimental

Table 2.5. Frequency of halothane susceptibility in some pig populations

(taken from Webb, 1981).

No. % halothane - Breed pigs positive

Duroc 248 0 American Yorkshire 225 0 Large White 1130 0 Hampshire 232 2

Landrace: Norwegian 576 5 Danish 1990 7 British 1538 11 Swiss 7480 13 French 127 17 Dutch - 4073 22 German 1251 68 Belgian 1260 86

Pietrain: French 335 28-68 German 266 87 Dutch 101 94

].

herds.

VII. PRACTICAL IMPLICATIONS.

The identification of the halothane locus with its major effects on

several economic traits undoubtedly has several practical implications.

From the point of view of research the discovery is useful for a better

interpretation of results from experiments concerned with traits whose

variances and covariances are partly determined by the frequency of this

gene. Heritabilities and genetic correlations will be affected as

described by Smith and Webb (1981); there could be non-linearity in the

regression offspring-mid parent and asymmetrical responses to

bidirectional selection (Robertson, 1977). Therefore, if neccesary it is

now possible to design experiments without the confounding effects of the

halothane gene.

From a breeding viewpoint the case for manipulating the gene will

finally depend on economical considerations. It could be convenient to

eliminate the gene or even to fix it in specialised sire lines with the

purpose of producing a heterozygous (Nn) commercial generation, as

discussed by Smith (1981 a), Smith and Webb (1981) and Webb (1981). In

any case, reliable information on the genotypic values for all economic

traits is still required.

In small breeding programmes it would not be very difficult to

manipulate the gene. Fixation should take a single generation of

selection. The time until elimination will vary according to the breeding

Page zz

plan: selection after test mating with homozygote recessives would be a

most effective method (Smith , 1981 a).

In breeding programmes of national proportions the opportunities for

manipulating the gene are less clear. If its existence is ignored and

there is selection in the direction of increased lean content there will

be an upward trend in the gene frequency towards fixation. The time to

fixation will depend mainly on the relative genotypic values, on the

selection intensity and on the fitness of suceptible animals. If there is

very low survival rate or culling of susceptible pigs or simultaneous

selection for traits like meat quality, an intermediate equilibrium

frequency could be reached. These problems were studied by Smith (1981

b). Again, to decide on an optimum strategy is an economic problem

depending mainly on the performances of the three genotypes and these have

not been firmly established yet. However, the implementation of such a

strategy at a national scale would be more difficult than in smaller

breeding programmes; some possibilities were discussed by Smith (1981 a).

VIII. QUESTIONS FOR FUTURE RESEARCH.

Although there has been a considerable amount of research in recent

years on the subject, several aspects still deserve further study; some

of them are of major importance from a practical point of view.

Thus, even though a single autosomal recessive model with variable

penetrance provides a reasonably good fit to the observed pattern of

inheritance, the meaning of the variable penetrance remains puzzling. The

possibility that there could be genetic variation in penetrance has to

rage U

be explored. Perhaps it would not be impossible to reduce the

susceptibility to stress by selection without losing some of the economic

advantages conferred by the n allele.

The genotypic values for all traits of economic importance should be

firmly established. Most of the estimates in the literature are from

analysis of data in field or testing station surveys. Although they can

provide initial indications there is still need for well planned

experiments. In this connection, it could be interesting to consider the

possibility of there being more than one normal allele having a range of

gene effects on production traits.

The problem of whether the effects attributed to the HAL locus arise

through pleiotropy or linkage disequilibria is still to be clarified. The

answer may provide the key for separating some of the beneficial from the

harmful effects.

Finally, although blood group testing can already be used to detect

the heterozygote genotype at the HAL locus (Jrgensen, 1981) the method

has some disadvantages, as discussed by Allen et al. (1980). Thus, the

development of new simple and reliable tests for identifying carriers of

the 11 allele is a challenging topic for future research.

CHAPTER 3. THE INHERITANCE OF HALOTHANE

SUSCEPTIBILITY IN PIGS -

INTRODUCTION

Many investigators have concluded that the reaction triggered by the

anaesthetic halothane in pigs is a recessive trait controlled by a single

autosomal locus (011ivier, Sellier and Monin, 1975, 1978; Minketna,

Elkelenboom and van Eldik, 1976; Smith and Bampton, 1977; McPhee, Takken

and DArcy, 1979; Mabry, Christian and Kuhlers, 1981). Other authors

have put forward alternative genetic explanations, including

single-dominant and two-locus modes of inheritance (Jones et al., 1972;

Williams et a].., 1975, 1978; Britt, Kallow and Endrenyi, 1978) -but have

not presented any formal genetic analysis supporting their conclusions.

There are no adequate studies as yet in the literature on the relative

merits of the single-recessive hypothesis tested under alternative

Mendelian models. However, taking into account the contrasting

interpretations mentioned above, and considering that the low penetrance

values in some studies (e.g. 011ivier et al., 1975, 1978) might indicate

a poor description of the events by the single-recessive model, there is a

case for more thorough hypothesis testing. This can be particularly

informative on data where it is not immediately obvious that a

single-recessive mode of inheritance provides the most adequate

explanation.

The purpose of this study was to test the validity of the

single-recessive hypothesis for mode of inheritance of halothane

susceptibility in pigs, within the framew )rk of (I) a single locus model

and (II) a model involving two epistatic loci. The models were fitted to

data from experimental Pietrain/Hampshire and British Landrace herds by

the method of maximum likelihood.

MATERIAL AND METHODS

Animals.

Halothane testing results from a synthetic population founded from

crosses of Pietrain and Hampshire and a British Landrace experimental

population were used in this study. All pigs received one 3-minute

halothane test at between 5 and 10 weeks of age as described by Webb and

Jordan (1978). Pigs developing a clear rigidity of the hind limbs within

the test period were scored as positive reactors; the rest were

classified as negative reactors.

The Pietrain/Hampshire data were presented by Smith and Bampton (1977)

who first analysed this material. Briefly, pigs from the third generation

of a randomly mated synthetic population containing 40 % Pietrain and 60 %

Hampshire genes were subjected to the halothane test. The population was

subsequently divided into two lines by mating mainly reactors with

reactors and non-reactors with non-reactors. The offspring from these

matings were also halothane tested. In contrast to Smith and Bamptons

investigation, only those families with known parental phenotypes were

included in the present study. The frequency of positive reactions

amongst parents was 0.33. The testing results are given in the Appendix.

The Landrace data were collected in an experimental population set up

by the Animal Breeding Research Organisation (ABRO) after a survey of the

incidence of halothane sensitivity in British nucleus herds, which

revealed an average frequency of positive reactors of 0.12 for this breed

(Webb, 1980). The animals constituting the parental group were purchased

from nine of the surveyed herds after being halothane tested on their

original farms. The frequency of positive reactors in this group was

0.48. Two lines were then formed, mating reactors with reactors and

non-reactors with non-reactors. The progeny from these matings were born

and halothane tested at ABRO. These data are also shown in the Appendix.

The Pietrain/Hampshire and the Landrace herds were kept on different

farms.

Models.

The single locus recessive (SLR) hypothesis was tested within the

framework of a general single-locus model where both the heterozygote and

one of the homozygous genotypes were allowed to react to the anaesthetic.

The SLR hypothesis was also tested within the framework of a two-locus

model involving a susceptibility locus and a suppressor locus. The

purpose of this model was to explain genetically part of the variation in

penetrance as observed under the SLR hypothesis. In all cases two

phenotypes were considered: reactor (R) and non-reactor (MR). The models

were fitted to the data by the method of maximum likelihood, following

Smith and Bamptons procedure (1977). This procedure will be described in

detail for the single-locus model and outlined for the two-locus model.

(i) Model 1. Single-Locus. The model requires two alleles: n, with

frequency q, and N, with frequency p (= 1 - q). Mating was at random in

the Pietrain/Hampshire population before the subdivision; therefore, for

the parental generation the expected genotypic frequencies and the

penetrances are

Genotype

MN Nn tin

Code (1) 1 2 3

2 Frequency p 2pq q 2

Penetrance 0 f4 f2

Some special cases under this model are f 1 = f = 1 (dominant,

completely penetrant) and f4 0, f2 1 (recessive, completely

penetrant). The model does not allow phenocopies.

The prior joint probabilities of parental phenotypes and genotypes (Q.

and Q.) are

Phenotype

R MR

Probability....

Genotype

NW 0 p2

Mn 2f4 pq 2(1-f4 )pq

nn f (1-f2 ) q

The probabilities of reactions among the progeny of the different

matings (P.. ) are conditional on the parental genotypes and are as shown

below

Sire (1)

NN Nn nn

NN 0

Dam (j) Nn f /2 (2f +f)/4 /2

nn f (f4 +f2 )/2 f2

The joint likelihood for a population with s sires, each mated to a

variable number of dams d, is given by

L =E (A -zd) T1 F, [(QjZ.

Lj P.

+ -Z))

()

(!)zJ )LJ - R u) 1} • .

where z = 1 if the parent is a reactor and z = 0 if it is a

non-reactor, the index k refers to the number of genotypes in the model, N

is the number of progeny from a particular mating and R is the number of

progeny reacting to halothane from that mating. Equation (1) is general

and holds for all models in this study. In the present case the

likelihood is a function (0) of three parameters L (q , f , f ).2.

In Landrace, the parental generation was sampled from the British

Landrace nucleus population. Although the frequency of haláthane positive

reactions in this population was 0.12 (Webb, 1980), roughly equal numbers

of positive and negative reactors were purchased for the foundation

generation of the experimental lines. For this reason the terms Q ;. and

Q. in equation (1) must now represent the prior probabilities of parental

genotypes conditional on phenotype. In contrast to Pietrain/Hainpshire

the probabilities Q. and Q. are conditional on phenotypes in all the A.

analyses of Landrace data throughout this study.

Also distinct from Pietrain/Hampshire, the parental Landrace group was

composed of pigs from nine different herds; therefore, the expected

genotypic frequencies are no longer represented by the Hardy-Weinberg

proportions. However, assuming equilibrium holds in the different

subpopulations, the expected parental genotypic frequencies are functions

of the mean () and the variance (V q ) of the gene frequency. Thus, taking

these facts into account, if the frequency of halothane positive reactions

is given by

F = 2f 4 (p q - V ) +

+ Vq )

the conditional probabilities of parental genotypes for the Single-Locus

model in Landrace are

Genotype Q. A. A.

NN 0 C p + Vq )/ (1 - F)

Nn 2f4 ( p q _ V q )/ F 2(1 - f ,1 )(- Vq )/ (1- F)

nn f2 ( + V )/ F (1 - f )( + V )/ (1 F)

The joint likelihood is now a function of four parameters: q, V1 , f4,

f. The SLR hypothesis is obtained if the restriction f 1 = 0 is imposed on

the model.

(ii) Model 2. Two—Locus. The first locus is assumed to determine

susceptibility to the anaesthetic and have two alleles: n with frequency

q and N with frequency p = 1 - q. The second locus is assumed to be a

suppressor locus, also with two alleles: S with frequency u and s with

frequency v = I - u. Under this model pigs require two copies of n at the

susceptibility locus and at least one S allele at the suppressor locus to

be positive reactors. A double dose of s will suppress the reaction in nn

pigs. Genotypes nnSS and nnSs are assumed to have the same penetrance

(f). The suppressor locus acts as a genetic device removing part of the

variation in penetrance as would be observed under the SLR hypothesis.

In general the two loci may be linked and the population may not be in

linkage equilibrium. Two types of double—heterozygotes must be

recognized: coupling (NSIns) and repulsion (NsInS). With random mating,

as in. Pletrain/Hampshire, and when linkage disequilibrium D, the

expected genotypic frequencies in the parental group and the corresponding

penetrances are shown in Table 3.1. The conditional probabilities of

reactor progeny given the parental genotypes can readily be computed.

Thus. for the mating NSIns x NSIns:

P = fO(2 —&)/4 55

where 0 is the recombination frequency. The joint likelihood is thus

a function of five parameters: q, v, D, 6 and f. It is possible to test

Table 3.1. Genotypic frequencies and penetrances in the two-locus model.

Genotype Code (i) Frequency Penetrance

NNSS 1 (pu + 0

NnSS 2 2(pu + D)(qu - D) 0

nnSS 3 (qu - f

NNSs 4 2(pu + D)(pv - D) 0

NStns 5 2(pu + D)(qv + D) 0

NsInS 6 2(pv - D)(qu - D) 0

nnSs 7 2(qu - D)(qv + D) f

NNss 8 (pv-D) 2 0

Nnss 9 2(pv - D)(qv + D) 0

10 (qv + 0

the hypothesis that the population is in linkage equilibrium CD = 0) and

that there is free recombination between the - two loci (6 = 0.5). After

such restrictions a simpler model is obtained where the joint likelihood

is a function of three parameters: q, v and f. This will be called the

Restricted Two-Locus model. The SLR hypothesis is obtained when the

restrictions v = D = 0 and 9 = 0.5 are imposed on the model.

In Landrace, where the parental generation was a mixture of

subpopulations, the genotypic frequencies can be approximately represented

by functions of the mean gene frequencies (lif and V), the variances of gene

frequencies (V q and V) and the covariance between allelic frequencies at

the two loci (Coy, ), after assuming equilibrium holds in the different

subpopulations. For example, the frequency of NNSS pigs is

I Z freq (NNSS) = E(p. U. ); A A.

after dropping a term involving fourth order moments of differences in

gene frequency

22 _2 _2 -- freq (NNSS) ii + i V + u V + 4 p u COV(qv)

Assuming free recombination between the two loci the joint likelihood

in Landrace is a function of six parameters: i, v , V 9 Vs,, Cov(qv) f

The SLR hypothesis is obtained when the restrictions v = = Cove v 0

are imposed on the model.

Computations.

A computer program was written to evaluate equation (1) for the

different models in this study. The likelihood surface was searched by

iteration within the parameter space; the maximum likelihood was thus

located and the co—ordinates of this point provided the ML estimates for

the different parameters.

All hypotheses were tested by means of the likelihood ratio MR)

criterion

LR=2 [ln (at )—ln(,,3)]

where in(o. ) and ln( 16 ) are the natural logarithms of the likelihood

maxima under the unrestricted and restricted models respectively. The LR

criterion was compared with a I distribution with n degrees of freedom, n

being the number of parameters on which restrictions were imposed in order

to define the null hypothesis.

Approximate confidence regions for some pairs of parameters in the

different models were estimated by constructing contour maps of constant

values ' on the log likelihood surface such that

2 [in (o ) - ] = 4 = 5.99, 4.60 and 3.22

where in( o. ) is the logarithm of the maximum likelihood and the

values correspond to the 0.05, 0.10 and 0.20 probability levels.

RESULTS

0.30 0.50

0 0 0

CU

..- -J

95

Page 34

Figure 3.1. Likelihood surface for the SLR hypothesis

in PietraiEL/Hampshire.

0 0 -c

—a

0.9(

0.90

Page 35

Figure 3.2. Likelihood surface for the SLR hypothesis in

Landrace. Variance of q (V q ) held at zero.

0.20

Table 3.2. Parameter estimates in Pietrain/Hampshire

under Model 1 (Single-Locus).

Gene frequency, q 0.61

Penetrance of Nn, f 4 0.00

Penetrance of nn, f2 0.91

Table 3.3. Parameter estimates and differences in log likelihood in

British Landrace under Model 1 (Single-Locus).

Single-locus SLR SLD model hypothesis hypothesis

Parameter f4 0 f1 =f2 0

Mean gene frequency, q 0.24 0.47 0.26

Variance of q, V 0.055 0.100 0.039

Penetrance of Nn, f4 0.22 - f 0.78 Penetrance of nn, f 0.91 0.67 't 0 . 78

LR criterion 17.22 15.57

Probability of greater 0.00003 0.00008

All likelihood surfaces scanned in the study exhibited a single

peak which, in general terms, was always fairly well defined. Figures 3.1

and 3.2 ilustrate typical likelihood surfaces in Pietrain/Hainpshire and in

Landrace respectively.

Model 1. Single-Locus: Table 3.2 shows the parameter estimates

under the Single-Locus model in Pietrain/Hampshire. The estimated

penetrance of the heterozygous genotype was 0.00, which is the value

assumed by the SLR hypothesis. Approximate confidence regions are shown

in Figures 3.3 and 3.4.

The results of the Single-Locus analysis in Landrace are summarized in

Table 3.3. The SLR hypothesis was rejected on the ?(.Z test (P<0.01)

indicating that the addition of a non-zero penetrance for the

heterozygotes made a significant improvement in the fit of the model to

these data. Approximate confidence regions for the two penetrances are

shown in Figure 3.5.The marked increase in V when moving from the general

Single-Locus model to the SLR hypothesis is to be noticed. This could be

interpreted as V conferring some flexibility to an intrinsically

inadequate hypothesis.

Model 2. Two-Locus: Table 3.4 summarizes the analysis under the

Two-Locus model in Pietrain/Hampshire. There was no indication of linkage

and the amount of linkage disequilibrium between the two loci was small

-about 20 % of the maximum disequilibrium possible. The inclusion of

these parameters did not improve significantly the fit of the model. The

SLR hypothesis was thus tested against the Restricted Two-Locus model;

0.7 0.8 0.9 1.0

0.12

0.10

0.08 C)

C) 0.06 C)

0

0.04

0.02

Page 38

Figure 3.3. Contours of constant log likelihood for the

Single—Locus model in Pietrain/Hazpshjre,

representing the approximate 0.05 ( —),

0.10 (— — —) and 0.20 ( ...... ) probability

levels. Gene frequency q held at 0.61.

Penetrance (f 2 )


Single-Locus model in Pietrain/Hampshire,

representing the approximate 0.05 ( ),

0.10 (— — —) and 0.20 ( ......) probability

levels. Penetrance of Nn ) held at zero.11

0.7

00

LI

Gj

.. :'

a- G.J

C)

C) 0.5

'..

0.4

0.7 0.8 0.9

1.0

Penetrance (f2)

Table 3.4. Parameter estimates and differences in log likelihood

in Pietrain/Hampshire under Model 2 (Two-Locus).

Parameter

Generalized two-locus model

Restricted two-locus

model D0, 9 0..5

SLR hypothesis

v0

Gene frequency, q 0.66 0.64 0.61

Gene frequency, v 0.50 0.38 -

Penetrance, f 1.00 0.97 0.91

Linkage disequilibrium, D -0.034 - -

Recombination frequency,O 0.50 - --

LR criterion 1.72 2.26 #

Probability of greater 0.42 0.13 #

# SLR hypothesis versus Restricted two-locus model.

Table 3.5. Parameter estimates and differences in log likelihood in

British Landrace under Model 2 (Two—Locus).

Restricted Two—Locus SLR SLD model hypothesis hypothesis

Parameter VVCOVqyçO c11, VqCO\qO

Mean gene frequency, 0.63 0.47 -

Variance of q, Vq 0.093 0.100 -

Mean gene frequency, 0.67 - 0.74

Variance of v, V 0.009 - 0.039

Covariance (Cov(v) ) —0.025 - --

Penetrance, f 0.81 0.67 0.78

LR criterion 9.83 8.19

Probability of greater 0.020 0.042

the LR criterion was 2.26 which is not a very conclusive result for a

variable. Figure 3.6 shows the confidence regions for the two gene

frequencies under the restricted Two-Locus model in Pietrain/Hampshire.

The results of the analysis under the Two-Locus model in Landrace are

shown in Table 3.5. After testing the SLR hypothesis the LR criterion was

9.83 which is statistically significant for a X.variable. Therefore, the

SLR hypothesis was also rejected under the Two-Locus setting (P<O.Ol).

DISCUSSION

The parameter estimates under the Single-Locus model in

Pietrain/Hampshire differed somewhat from those obtained by Smith and

Bampton (1977). The discrepancy could be due to the fact that only a

subset of their data was used in the present study. In agreement with

Smith and Bampton the likelihood was maximised when the penetrance of the

heterozygotes was equal to zero. These results do not disprove the SLR

hypothesis. A different picture emerges from the Single-Locus analysis in

Landrace. The parameter estimates indicate that about a quarter of the

heterozygotes were positive reactors after the halothane test. An

inspection of the Landrace data does not reveal an easy agreement with the

SLR hypoithesis. On the one hand there is a deficiency of segregating

litters among the negative matings while, on the other hand, there is

heterogeneity in the segregation ratios among the progeny from the

positive matings, with several families exhibiting what would appear to be

very low penetrance values. No such heterogeneity was observed among the

positive matings in Pietrain/Hampshire.

?agc 43


Single-Locus model in Landrace, representing

the approximate 0.05 ( ), 0.10 (— — — )

and 0.20 (......) probability levels. Mean

gene frequency q held at 0.24; variance of

gene frequency (V q ) held at zero.

In

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II I , II I , I , I / S

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0.5 0.6 0.7 0.8 0.9

Penetrance )


Restricted Two-Locus model in Pietrain/

Hampshire, representing the approximate 0,05

( ), 0.10 (— - —) and 0.20 (......)

probability levels. Penetrance (f) held at 0.97.

0.8

0.7

> 0.6

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Gene frequency (q)

It is possible to test the hypothesis that both heterozygous and

homozygous pigs did react to the anaesthetic with equal penetrance; this

amounts to test a single-dominant (SLD) hypothesis. As shown in Table

3.3, the SLD hypothesis was rejected on the 2C test result. Thus, in

contrast to Pietrain/Hainpshire, there appears to be a gene dosage effect

in Landrace whereby carriers of a single copy of the susceptibility

allele would have a smaller (though non-zero) penetrance than carriers of

two copies of such allele. The reasons for this difference between

Pietrain/Hampshire and British Landrace are unknown. As the two

populations were kept on different farms there could have been differences

in relevant environmental circumstances making some of the heterozygous

Landrace pigs susceptible to the anaesthetic. However, as little is known

about such environmental factors it is difficult to speculate on how a

difference might arise. It is possible, though, to conceive a number of

genetic explanations. Most of them -such as the presence of more than two

alleles at the susceptibility locus or the breeds differing in modifier or

suppressor gene frequencies- require a broadening of the simple

single-biallelic model favoured so far. The Two-Locus model in the

present study represents one such explanation -not necessarily the most

adequate, of course.

Halothane susceptibility thus resembles the double muscle trait in

catiie In that the mode of Inheritance seems to differ between breeds

Under a single-locus hypothesis the double muscle' trait appears to be

recessive in some breeds and dominant in others (Menissier, 1982). Other

similarities between these two traits have already been pointed out

(011ivier, 1980).

Although not conclusive, the results of the analysis under the

Two-Locus setting in Pietrain/Hampshire suggest that a model removing

genetically part of the variation in penetrance could explain the

observations better than a single locus with penetrance as a purely

environmental parameter. A mixed model -a major locus, polygenic

variation and environmental effects all contributing to an underlying

liability scale with a threshold determining susceptibility (Morton and

MacLean, 1974)- could perhaps perform the task more flexibly. However, it

is unlikely that in the present circumstances it would have fitted the

data significantly better than the simple two-locus model.

The Two-Locus analysis in Landrace also rejected the SLR hypothesis.

It is possible to test the hypothesis that

q = 1 and Vq = Cov(V) = 0;

after such restrictions the Two-Locus model yields the single-dominant

(SLD) hypothesis. The LR criterion indicated that the Two-Locus model

also fitted the data better than the SLD hypothesis (P<0.05). The maximum

likelihood obtained under the general Single-Locus model was higher than

that obtained under the Two-Locus ode1 It was not possible to test both

models as hypothesis in the same analysis. A general model allowing such

a test would have been unwieldy given the structure of the Landrace

population.

In summary the SLR hypothesis, favoured so far as the mode of

inheritance of halothane susceptibility in pigs, could not be conclusively

disproved in Pietrain/Hampshire although there was a suggestion that part

of the variation in penetrance could be genetically determined. The SLR

hypothesis was clearly rejected as the mode of inheritance in British

Landrace. It is important to emphazise the fact that the Landrace parents

were tested in their original farms; the varying environmental conditions

might have increased the probability of misclassifying the reactions. The

lack of matings between reactors and non—reactors and the mixture that

constituted the parental group in this breed should also be eniphazised.

Because of the latter the probability models describing the population

required parameters such as variances and covariance of gene frequencies;

conclusions of general interest were thus conditional on the value of

nuisance parameters in the models. Taking into account all these

deficiencies the present findings should be considered as preliminary

indications that the generally accepted single and strictly recessive mode

of inheritance may not be adequate for the British Landrace breed. Should

these findings be confirmed, a unified explanation of the observations in

different breeds will probably require a more comprehensive genetic model

than a single—biallelic locus. Such new developments, however, would not

detract from the usefulness of the halothane test as a practical screening

tool for reducing the incidence of Porcine Stress Syndrome in pig

populations by means of selection.

CHAPTER 4. STUDIES ON THE TIME OF ONSET OF REACTION

TO HALOTHANE ANAESTHESIA

INTRODUCTION

It was shown in Chapter 3 that a model involving a susceptibility

locus and a genetic device accounting for part of the variation in

penetrance could describe the inheritance of reaction to halothane

anaesthesia better than the widely accepted single-recessive model, where

penetrance is assumed to have an entirely environmental determination.

The time of onset of reaction is a concrete quantity which could be

related to the concept of penetrance as follows: the test duration being

limited, those pigs having longer reaction-time would be classified as

negative reactors; penetrance would thus be lowered. The question then

arises as to what proportion of the variation in reaction-time is under

genetic control; if it exists, such genetic variation should be regarded

as modifying the susceptibility status determined by the main locus (or

loci). The purpose of this study was to estimate phenotypic and genetic

parameters of the time taken to react positively to halothane anaesthesia

by Pietrain/Hampshire and Landrace pigs.


Animals.

The data for these studies were collected from a Pietrain/Hampshire

(PTH) and a British Landrace experimental line, both selected for

Increased susceptibility to halothane at the Animal Breeding Research

Organisation (ABRO). The PTH line was selected for six years, with some

overlapping of generations. Two non-overlapping generations of selection

were carried out in the Landrace line (see Webb, 1981). The two lines

were kept on different farms. All pigs received a halothane test at about

eight weeks of age, as described by Webb and Jordan (1978). The duration

of the test was 3 minutes, except In the second Landrace generation when

it was extended to 5 minutes. The pigs were classified as positive,

doubtful and negative reactors; only the former were selected as breeding

stock. All positive reactions recorded in the two lines provided

information for the present studies. The trait under study,

reaction-time, was defined as the time elapsed from the start of

anaesthetic administration until the animal exhibited definite signs of

halothane susceptibility: muscle rigidity at the hind limbs, after which

administration of anaesthesia was interrupted. These data were used in an

attempt to estimate the heritability of reaction-time at eight weeks of

age. Records from 403 PTH and 291 British Landrace halothane positive

pigs. from the two selection lines were available for this purpose.

In order to assess the effect of age on halothane susceptibility three

trials were carried out at ABRO. The design and results of these trials

will be described in Chapter 5; only relevant information will be given

here. Trial 1 comprised the progeny of the fourth year of matings of the

PTH susceptible line. In addition to the test at 8 weeks these pigs

received two previous 3-minute tests, at about 3 and 5 weeks. A similar

trial (Trial 2) was carried out with the progeny of the first generation

of the Landrace susceptible line. Trial 3 was performed on the second

Landrace generation: all pigs each received four 5-minute tests, at about

3, 5, 7 and 9 weeks of age. As the animals in these trials received

repeated tests the results allowed estimation of repeatability of

reaction-time. The number of pigs at each test, their average age and

weight, and the number of positive reactors in Trials 1, 2 and 3 are shown

in Table 4.1.

Statistical analyses.

Analysis of heritability. Least squares analyses of variance were

carried out, using the computer program LSML76 (Harvey, 1977).

Exploratory analyses showed that the day of testing had very important

effects on reaction-time. In each breed, separate hierarchical analysis

were then performed on the log 40

reaction-time, fitting a model involving

test-day, sires, dams, sex and weight (covariate). It was assumed that

sires were nested within test-day although, in fact, about 30 % of them

appeared twice; those appearances were counted as different sires. Table

4.2 shows the degrees of freedom from the analyses of variance and the

expectation for the sires mean squares. Standard errors for the

heritabilities were estimated as suggested by Woolf (cited by Faicuuer,

1963).

Analysis of repeatability. The time of onset of all positive

reactions recorded in Trials 1, 2 and 3 (Table 4.1) were analysed by least

squares analysis of variance, using the LSML76 program. Each trial was

Table 4.1. The number of pigs at each test, their average age (SD),

weight (SD) and the number of positive reactors, in

Trials 1, 2 and 3.

Test

Trial Trait 1 2 3 4

No. pigs 66 66 66 -

Trial I Age (days) 18(1.8) 35(1.8) 53(1.8) -

PTH Weight (kg) 4.7(2.4) 8.2(2.4) 14.3(2.4) -

(3—mm) No. positive 27 42 55 -

No. pigs 201 198 198 -

Trial 2 Age (days) 21(2.5) 35(2.5) 56(2.5) -

Landrace Weight (kg) 5.3(2.4) 8.7(2.4) 15.4(2.4) -

(3—mm) No. positive 39 123 117 -

No. pigs 253 249 246 244 Trial 3 Age (days) 21(0.9) 35(0.9) 49(0.9) 63(0.9)

Landrace Weight (kg) 5.7(2.7) 9.6(2.7) 15.2(2.7) 19.7(2.6) (5—mm) No. positive 161 215 205 220

It is"

Table 4.2. Degrees of freedom from the analyses of variance

for estimating heritability of reaction-time in PTH

and in Landrace.

Breed:

Source of variation PTH Landrace

Test-days 36 15

Sires/Test days * 41 25

Dams/Sires 13 8

Sex 1 1

Regressions

Weight-linear I 1

Weight-quadratic 1 1

Remainder 309 239

* Expected value of sires mean squares: t 2 2

PTH: + 3.7 T3 + 4.3 TS

Landrace: + 4.7 + 5.2

where T , and 0 are the within-litter, w D S

dams and sires variance components.

analysed separately. A simple analysis was done first to examine the

effects of test and sex on reaction—time. The statistical model was

Y = ,U. + T. + S. J U + (TS).. + e..

4j K A.

where Y.. is the reaction—time of the kth pig of the jth sex (male or

female) in the ith test (1, 2, 3 or 4).

In a second analysis a model involving a term for individual pigs was

fitted to different subsets of data from Trials 1, 2 and 3 in order to

2 '2. estimate the variances between () and within (cf) pigs. Repeatability

was estimated as

2.2 '2.

t =T /( + ¶, )

with standard error as suggested by Swiger et al (1964). The model

used was

2. Y )A +S

+/W ~/w ~ e.

.1 j1'c .2. Kt

where Y j is the log 40

of the kth reaction—time of the kth pig,

within the jth sex, having a live weight W. As Trials 1 and 2 included

three tests they allowed estimation of t between 3 different pairs of

tests, as well as among all three tests. Trial 4, comprising four tests,

allowed estimation of t between 6 different pairs of tests and 4 different

triplets, as well as among all 4 tests. The coefficients k for the

2. variance component , in the expectation of the mean squares for P,

ranged from 1.25 to 1.82 when t w is estimated between pairs of tests;

from 1.91. to 2.82 when triplets were used; k = 3.27 when t was estimted

among all four tests in Trial 3.

RESULTS

Figures 4.1 and 4.2 show the distributions of reaction-time at about

eight weeks of age in PTH and in Landrace respectively. In both cases the

distributions were skewed to the left. The logarithmic transformation

reduced the skewness appreciably.

(i) Analysis of heritability. The analyses of variance showed that

the day of testing was a most important source of variation accounting, on

average, for some 30 Z of the total sums of squares. In both breeds, but

particularly in Landrace, the dam component (0) was larger than the sire

component (cT z ). This could be due to dominant, epistatic and non-genetic

maternal variances. Table 4.3 shows the three estimates of h2 . In

neither PTH or Landrace were the differences between sires statistically

significant. Differences between dams were significant in PTH (P<0.01).

It must be pointed Out, however, that the statistical model was more

adequate for estimating variance components than for testing hypotheses.

The assumption that sires were nested within test-days led to counting

some of the sires twice, producing the wrong degrees of freedom for this

effect. There is also uncertainty about the importance of possible sire x

test-day interactions.

W

60

50

40

30

20

10

Page 55

Figure 4.1.. Distribution of. time of onset of reaction to

halothane'at eight weeks of age in Pietrai,V

Hampshire pigs.

30 60 90 120 150,180

Reaction time (sec)

Mean = 84 sec

Standard deviation = 40.3 sec

Coefficient of skewness = 1.45

coefficient of kurtosis = 2.00

C, .0 ou u IZO 150 180 210 240 270 300

Reaction time (sec)

Page 56

Figure 4.2. Distribution of time of onset of reaction to halothane at about eight weeks of age in (a) the first and (b) the second generation Landrace.

(a) 50

40

30

20

10

0•

30 60 90 120 150 180

Reaction time (sec)

(b)

50

40

30

20

10

(a) Mean = 117 sec Standard deviation = 40.8 sec -Coefficient-of skewness = 0.06 Coefficient of kurtosis = -1.14

(b) Mean = 145 sec Standard deviation = 65.3 sec Coefficient of skewness = 1.14 Coefficient of kurtosis = 0.55

Table 4.3. Heritability estimates (SE) of the time taken to react

to halothane anaesthesia in Pietrain/Hampshire and in

British Landrace pigs.

Heritability estimate

2. 2. Breed h

2 s h hs+D

Pietrain/Hampshire 0.65 (0.592) 0.90 (0.527) 0.77 (0.144)

British Landrace 0.12 (0.582) 0.45 (0.694) 0.28 (0.277)

(ii) Analysis of repeatability. The results from the analyses of

effects of test and sex on reaction—time indicated that these were not

important sources of variation in Trial 1. In Trials 2 and 3, however,

the tests were a significant source of variation; in addition, females in

Trial 3 reacted faster than males. There were no significant test x sex

interactions. These results are shown in Table 4.4. There seems to be a

difference between trials, Trial 3 exhibiting longer reaction—times than

the other two. There also seems to be a trial .x test interaction, as the

pattern of reaction time lengthening with age which was observed in Trials

I and 2 was not observed in Trial 3. Since the trials were analysed

separately the statistical significance of these differences could not be

tested.

The reduction in total sums of squares after fitiig the term

(pigs within sexes) averaged 69 % when analysing pairs of test and 50 %

when dealing with triplets, over the three trials. Table 4.5 shows the

different estimates of repeatability in the three trials. In general

terms the repeatabilities were somewhat lower in Trial 3 than in the other

Table 4.4. Least squares means (X) and standard deviations (SD) of

reaction-time (seconds) for the four tests and the two

sexes, and F ratios from the analyes of variance, in

Trials 1, 2 and 3.

Trial I Trial 2 Trial 3 PTH (3-mm) Landrace (3-mitt) Landrace (5-mitt)

Source X SD X SD X SD

Test number

1 75 39.0 85 41.2 163 64.7 2 84 39.5 101 38.8 106 64.5 3 96 40.0 118 38.9 138 64.4 4 - - - - 146 63.8

F-ratio: 2.70 11.19** 26.15**

Sex

Males 85 40.2 99 47.8 149 65.0 Females 85 40.7 104 43.1 128 65.0

F-ratio: 0.00 0.98 21.58**

** P < 0.01.

1-2-4 1-3-4

1-2-3-4

0.11 0.050 0.11 0.18 .051 0.18

0.15 0.038 0.15

Page 59

Table 4.5. Repeatability (t) of reaction time estimated

over different tests, in Trials 1, 2 and 3.

Trial 1 Trial 2 Trial 3 Pooled Test

combination t SE ̂t SE t SE t

1-2 0.40 0.191 0.44 0.135 0.34 0.074 0.36 2-3 0.39 0.155 0.40 0.087 0.30 0.070 0.33 3-4 - - - - 0.20 0.073 0.20

1-3 0.26 0.216 0.44 0.145 0.24 0.084 0.27 2-4 - - - - 0.18 0.072 0.18

1-4 - - - - 0.04 0.091 0.04

1-2-3

0.29 0.080 0.39 0.075 0.20 0.052 0.25

2-3-4 - - - - 0.23 0.047 0.23'

two trials. There was also a decreasing trend in repeatability, as

estimated between pairs of tests, as the time interval between tests

A increased. The t values ranged from about 0.30 when this interval was

about two weeks to 0.04 when it was six weeks. A similar trend can be

A observed among the t values estimated from triplets.

DISCUSSION

It was shown in a previous section that a model accounting genetically

for variation in penetrance might fit the inheritance of halothane

susceptibility better than a single-locus model with penetrance as a

purely environmental parameter. The main purpose of the present study was

to detect signs of genetic variation in the time susceptible pigs take to

react to halothane, associating this concrete quantity with the concept of

penetrance. Figure 4.3 illustrates how, with a given test duration,

differences in the mean and variance, of reaction-time between two

hypothetical populations, a and b, might cause a proportion of susceptible

subjects to be classified as negative reactors.

The number and size of the sibships and the shape of the frequency

distributions discouraged the search for a major gene affecting

reaction-time. Such analysis was further discouraged by the possibility

that any multimodality in the frequency distributions (Merat, 1968) might

have been blurred by a too short halothane test. Instead, the analysis

was directed towards detecting differences between half-sib groups and

estimating repeatabilities and heritabilities, even though the structure

and amount of data were far from ideal for these purposes. Furthermore,

Figure 43, The association between time of onset of reaction and penetrance of halothane susceptible genotypes

Beginning End of Reaction of test test time

High penetrance population

Low penetrance population

if the test was shorter than the range of reaction-times, there could have

been selection for fast reactions among the parents (which were selected

for susceptibility on a 3-minute basis) and truncation of the phenotypic

frequency distributions among the subjects of these studies. This could

have biased the heritability and repeatability estimates, probably

downwards, while increasing their standard errors (Robertson, 1977).

As for the possibility of truncation, when the test was extended to 5

minutes, in Trial 3, about 18 % of all positive reactions occurred after

the third minute, both at 7 and at 9 weeks of age (Figure 4.4). The

longer mean reaction-time in Trial 3 was, In fact, a consequence of the

longer halothane test. Thus, a 3-minute test could have been too short

for the purposes of this study. Methods of estimation on censored data

are available; Maximum Likelihood estimation would be appropriate for the

type of censoring in the present data. However, a distribution function

has to be assumed and there is no evidence which would justify the

adoption of any particular one. Other methods of estimation are very

troublesome (Kendall and Stuart, 1979; vol 2, pp 551-556). Therefore,

conventional estimation methods were used ignoring the problem of

censoring; the study, however, was intended as a tentative, preliminary

examination.

The fact that the herItabilIty estimates were all higher than the

repeatability estimates in PTH is a somewhat anomalous result. Perhaps

the requirement that the trait at different ages remains genetically the

same was not met, even though reaction-time was not greatly affected by

the different tests in this breed. However, the heritabilities were

estimated with very low precision and the figures in Table 4.3 are

50

40

30

20

10

0

(b)

C

50

40

30

20

10

0

(a)

A'

Page 63

Figure 4.4. Distribution of time of onset of reaction to halcthane at seven (a) and nine (b) weeks of ace in British Landrace pigs (Trial 3).

1 2 3 4

1 2 3 4 5

Reaction time (mm)

Reaction time (mm)

reported for illustration more than anything. The analyses failed to

detect significant differences among sires, both in PTH and in Landrace.

The only results that could serve as indication of genetic determination

are the repeatabilities, which were estimated with better precision;

however, they merely represent an upper limit to the degree of genetic

determination of a trait. The estimates were in broad agreement with a

value of 0.3 reported by Webb and Jordan (1979).

This study thus failed to produce satisfactory evidence of genetic

variation in the time of onset of reaction to halothane, which should not

be surprising in view of the limited information available. Further

research is necessary, on what could be an Important aspect of halothane

susceptibility. The possibility of censoring the data with a test too

short must be taken into account. There are reports of halothane positive

reactions recorded, on average, after 8 and 28 minutes of anaesthesia, in

two different pig crosses (Britt, Kallow and Endrenyi, 1978) and even

after 40 minutes in Poland China pigs (Allen, 1980; Jones et al., 1972).

This suggests that a test longer than 5 minutes could be necessary for a

proper study of the genetics of reaction-time and for a better

understanding of halothane susceptibility.

Reaction-time could enter in different ways in models for the

nhertance of halothane susceptIbIlIty, as Illusttated by the two

following examples. Figure 4.5 shows how the effects depicted in Figure

4.3 might be caused by gene subtitution at a single locus. The variance

in population a is assumed to be environmental. The increases in mean and

variance in population b are due to segregation, at intermediate

frequency, of a mutant allele. This allele increases reaction-time in a

raL .1 Ofl

time nn nn tin 55 55 ss

Page 65

Figure 1e5 • A model for the genetic control of penetrance of a halothane susceptible genotype (nn) by a single locus with two alleles : S and s, affecting reaction-time (see text).

Beginning End of of test test

Population a : frequency (s) = 0 Population b : frequency (s) = 0.5

strictly additive fashion but, given the test duration, it appears to

suppress the reaction in a recessive way. Such a model is not entirely

different from the two-locus model used previously to study the mode of

inheritance of halothane susceptibility. The extension to polygenic

control of reaction-time is an obvious development.

The second example is sketched in Figure 4.6. As reported previously,

under a single locus model susceptibility was strictly recessive in PTH

while one homozygote and about a half of the heterozygotes appeared to be

susceptible in Landrace. Breed differences in modifier gene frequencies,

or multiple allelism at the susceptibility locus, were mentioned as

hypothetical genetic explanations. Figure 4.6 shows how they could be

expressed in terms of reaction time. All pigs would, sooner or later,

react to the anaesthetic. A major locus controls the time of onset of

reaction. The breeds differ in modifier frequencies (N is modified to N)

or in allelic frequencies at the reaction-time locus. Of course, the

models sketched in Figures 4.5 and 4.6 can be combined, and other,

different models are possible. A feature of these models is that the mode

of inheritance of susceptibility is an artifact of the test duration.

Thus, it could he important to examine properly the role of the time of

onset of reaction in the halothane susceptibility phenomenon, for which

halothane tests longer than 5 minutes may be required. The findings in

this s tudy that of _.cc_.____i b 1. LL '¼ r

weight) and sex of the pigs, and that the day of test can be a very

Important source of random variation might help in the planning of

experiments and data collections for future studies.

Figure 4.6. A model for the inheritance of halothane susceptibility

based on the time of onset of reaction (see text).

'Reaction time' locus, with 3 alleles : n, N, N '

Breed (- ...... ) :n and N ore ed 2 ( ) : nand N'

Ion

00

0' Beoinnlng Did of orteit test

CHAPTER 5. THE EFFECT OF AGE ON HALOTHANE SUSCEPTIBILITY

INTRODUCTION

The diagnosis of Porcine Stress Susceptibility by means of the

halothane test has been done at ages ranging broadly from 20 to 100 days

(Webb, 1980). From a practical point of view it may be convenient to

carry out the test at the youngest possible age, as pigs are then easier

to handle. However, since many traits cannot be observed before a certain

age of onset, a point of some interest is whether the incidence of

halothane susceptibility changes with the age of the animals. If this is

the case it might be necessary to establish a suitable age for testing in

practice. From a research viewpoint, appropriate allowances for an

age—dependent penetrance might have to be made when studying the mode of

inheritance of susceptibility in pigs of varying ages. It seems

important, therefore, to determine whether there are changes in the

incidence of susceptibility with advancing age. The purpose of this study

was to assess the effects of age on the frequency of halothane

susceptibility in pigs which were repeatedly exposed to the anaesthetic.


Experimental.

Page b5

Four trials were carried out; Trial 1 comprised offspring from the

third batch of matings of a Pietrain/Hainpshire (PTH) line selected for

halothane susceptibility at the Animal Breeding Research Organisation

(ABRO). All pigs were each given three halothane tests (I, II and III) at

about 3, 5 and 8 weeks of age. A similar trial (Trial 2) was done on

offspring from the first generation of a British Landrace line selected

for halothane susceptibility at ABRO. Trial 3 comprised pigs from the

second generation of the halothane susceptible Landrace line; all animals

received four halothane tests (I, II, III and IV) at about 3, 5, 7 and 9

weeks of age. The three trials were carried out in different years. The

PTH and the Landrace lines were kept on different farms; some details

about the two lines were given by Webb (1981) and a fuller description of

the PITH line will be given later in this thesis.

As animals in trials 1, 2 and 3 received repeated tests, a

conditioning effect of previous tests on subsequent ones might be

hypothesized. For example, it is conceivable that the probability of

positive reactions among pigs receiving their first test at a given age is

higher (lower) than that among pigs which, after previous exposure to

halothane, have become more tolerant (sensitive) to it. The hypothesis of

a conditioning effect was tested in Trial 4; pigs from the PTH line

were randomly divided among three treatments: (A) one test, at 8 weeks;

(B) two tests, at 5 and 8 weeks or (C) three tests, at 3, 5 and 8 weeks of

age. Trial 4 was replicated over two different years and comprised

offspring from the fourth and fifth batches of matings of the PTR

susceptible line.

The halothane testing procedure was described by Webb and Jordan

(1978). Anaesthesia was induced with 4 to 8 % halothane concentration in

oxygen (2-3 litres/minute) and maintained with a 0.5 to 2.0 % halothane

concentration. In all trials the test duration was 3 minutes except in

Trial 3, when it was extended to 5 minutes. The pigs were scored as

positive reactors (HP) when clear rigidity of the hind limbs was observed,

and negative reactors (RN) when they remained relaxed throughout the test.

Those pigs for which a clear diagnosis was not possible were scored as

doubtful (HD). Table 5.1 shows the distribution of pigs in the four

trials; Table 5.2 shows the average age and weight of the pigs at each

test. All pigs were offspring of HP x HP matings.

Statistical analysis.

The method of maximum likelihood was used to estimate the effects of

test and other variables on the probability of obtaining HP reactions.

Each observation y4 , y1 ,...,y was considered a binary random variableTL

taking value I if the individual was HP and 0 otherwise (i.e. RD or RN),

with probability

Pr(y. = 1) p. , !. 1, 2,..., ii. .4.

A transformation was required to represent the probabilities p. by a

linear function of r explanatory variables so that, on an underlying

scale

Table 5.1. The total number of pigs, and of HP and RD reactions,

by test and sex in trials 1, 2 and 3, and by test

and treatment in Trial 4.

Trial 1 Trial 2 Trial 3 Trial 4

Test Reaction d' - 2 0 2 A B C

I All 34 32 105 96 130 123 - -- 71 HP 14 1`3 13 26 76 85 -- - 51 HD 4 8 8 9 9 10 - -- 3

II All 34 32 104 94 128 121 - 50 70 HP 24 18 56 67 108 106 -- 39 42 HD 1 6 5 6 6 3 -- 4 3

III All 34 32 104 94 125 121 54 48 64 HP 24 28 49 68 99 106 37 54 61 RD 8 2 8 8 6 7 8 4 3

IV All -- -- -- -- 123 121 - -- -

HP - -- - -- 105 115 - - -

RD - -- -- -- 4 2 - - -

Table 5.2. The average age (days) and weight (kg) at each test

in all trials (standard errors in brackets).

Trials 1 0 2 and 4 (pooled) Trial 3

Test Age Weight Age Weight

I 19 (2.2) 5.1 (2.5) 21 (09) 5.7

II 35 (2.1) 8.6 (2.5) 35 (0.9) 9.6 (2.6)

III 54 (2.0) 15.2 (2.6) 49 (0.9) 15.2 (2.6)

IV - --- - 63 (0.9) 19.7 (2.6)

where the x. are the values of the explanatory variables for the ith xj

observation. The logistic transformation was chosen, which defines the

logit of p. as

L ln(p. 1(1 - P ;. ))

(Finney, 1970), so

p.e /(l+e )

As the logit scale ranges from - oG to + oO it allows the fitting of

linear models without any restriction on the parameters; furthermore, the

logistic probability density function

& f(&)e /(1+e )

is symmetric about 0 and, as shown in Figure 5.1, very similar to a

normal distribution.

The individual observations y were assumed to come from a binomial

rage I.)

distribution; the likelihood of the ith observation was therefore

Yi 4- y L.p (i — p.)

4.

and the likelihood for all observations was

11.

L =

ri L. A

h.l

Maximum likelihood estimates (MLE) of the parameters in 0 were found

by maximizing 2 T 9 the natural logarithm of L T9 with respect to ,M. and 1 J

(j = 1, 2,..., r). This was done with the computer program GLIM (Baker and

Nelder, 1978). GLIM uses an iterative algorithm, described by Nelder and

Wedderburn (1972), for finding the MLE.

All null hypothesis about sets of parameters in the linear models were

tested by means of the likelihood ratio criterion

LR = -7nax .4

where and are the log likelihoods maxima under models 1

and 2 respectively. Model 2 is nested within model 1 and soecifie

restrictions on the value of some parameters are imposed upon it; such

restrictions represent a particular null hypothesis. The LR criterion was

2. compared with a distributionwith d degrees of freedom; d was given by

the difference between the number of parameters estimated by the two

models.

Frequency

0.3

0.2

0.1

0

- -. -' - I 0 1 2 3 4

Figure! 5.1. The Logistic and Normal distributions: both with Al

0 and (T. (IT /3) 2.

Value of e

00

rage i

The statistical significance of several explanatory variables was

tested by fitting a sequence of nested models. The fullest model for

trials 1 and 2 was

O..+ TL + Sj + (TxS). + P + eKe

where ,u is the general mean, T. is the effect of the ith test (I, II

or III), Si is the effect of the jth sex (male or female), P. is the

effect of the kth pig within the jth sex and eKe is a random error

associated with the £th observation. The hypothesis testing procedure for

trials 1 and 2 is shown in Table 5.3.

Because of the limitation in the number of parameters that may be

estimated with GLIM it was not possible to include the set of parameters

(pigs within sexes) in the analysis of Trial 3. For the rest, the

fullest model fitted to these data was similar to that of trials I and 2;

T , the effect of the ith test was for I = 1, ..., 4.

The fullest model fitted to the data from Trial 4 was

0 T+S + Tr 0 + + (TxS),, + (TxTr)0 + (SxTr) 70 + Tnnoq M 71

e m-n b6

where T Tnis the effect of the mth test (I, II or III), S is the

effect of the nth sex, Tr0 is the effect of the oth treatment (A, B or C)

Table 5.3. Hypothesis testing procedure in trials 1 and 2

(for explanation of symbols see text).

Hypothesis

S = S t = 0

T 1 = T ;L = T 3 = 0

MS) 44

=,...,(TxS)3 = 0

PA = P =0

4 2. '' K.

LR

2[ ()c, T, S ) - (,Li, T )]

2[. (i&, T, S, P ) — 1(,s., S, P )]

2[ (full model) -i(,Lc., T, S, P)]

2[. (full model) - 2çP- T, S, TxS)]

Table 5.4. Hypothesis testing procedure in Trial 4

(for explanation of symbols see text).

Hypothesis

S4 = S 2 = 0

T = T 2. = T 3 =0

(TxS)44 0

Tr4 = Tr Tr = 0

(TxTr)44 =""' (TxTr) 33 0

,..., (SxTr) 3 = 0 (SxTr)4 =

Y .4 = Y 2 =0

IN 2['(,z,T,S,Tr,Y,TxTr) - i(,k,T,Tr,Y,TxTr)1

2[2 ()&,T,S,Tr,Y,SxTr) - j(,L(.,S,Tr,Y,SxTr)]

2[l (full model) - ,T,S,Tr,Y,TxTr,SxTr)]

2[ (,M.,T,S,Tr,Y,TXS) - 1( 1L,T,S,Y,TxS)]

2[J(full model) -(L&.,T,S, Tr, Y,ThS,SxTr)]

2[ 2 (full model) - (,LT,S,Tr,Y,TxS,TxTr)]

2[ .(full model) - 2(,14,T,S,Tr,TxS,TxTr,SXTr)]

and Y is the effect of the pth year (1. or 2). The hypothesis testing

procedure is shown in Table 5.4. Again, it was not possible to fit the

set of parameters representing the effects of individual pigs because

their number exceeded the capabilities of the GLIM program.

The same statistical analysis was repeated separately for the

probability of RD (versus HP or HN) and of HP+}ID (versus HN) reactions.

The results of a preliminary analysis on data from trials 1 and 4 were

presented earlier by Webb (1980, 1981).

RESULTS

The numbers of HP and HD reactions recorded in the four trials are

shown in Table 5.1. The results from the statistical analyses are

summarised in Tables 5.5 to 5.10. A feature of all trials is that the age

effect was assessed by repeating tests on the same pigs. As mentioned

earlier, the effects of the individual pigs were disregarded in trials 3

and 4 because of limitations in the number of parameters that could be

estimated. However, if these effects were important the LR tests of age

effects need not be- equal to those that could have been obtained had the

models included the parameters for individual pigs. Statistically,

thcrcfore, the test of age effects in trials 3 and 4 is conservative.

Effects of previous tests on subsequent ones.

It is convenient to present the results of Trial 4 in the first place.

rage '0

Table 5.5 shows the ML probability estimates of HP and HD reactions for

the three treatments. As shown in Table 5.6 there were no significant

effects of the treatments on the probabilities of either type of reaction.

The effects of test and sex on HP reactions, however, were statistically

significant.

If the treatments are ignored the data from trials 1 and 4 can be

conveniently pooled and re—analysed, as both were carried out on the same

type of pigs under the same conditions. The largest model for this

analysis was the one used in the analysis of Trial 3, to which .a term for

the effects of the different years (y, m = 1, 2 or 3) was added.

HP reactions.

Table 5.7 shows that the probability of this type of reaction

increased with age in all trials; these changes were always statistically

significant (Table 5.8). With the exception of Trial 1, all the results

indicate that the frequency of HP was significantly lower in males than in

females; the test x sex interaction, however, was significant in PTH pigs

(Table 5.8). Finally, the analyses of trials 1 and 2 revealed that the

pattern of halothane reactivity was significantly affected by the

individual pigs.

HD reactions.

Table 5.9 shows the estimated probabilities of doubtful reactions for

Table 5.5. Estimated probabilities (p) of HP and HD reactions

for the different tests and treatments in Trial 4.

Treatment

A B C

Test HP RD HP HD HP RD

I -- - -- -- 0.52 0.10

II -- -- 0.77 0.08 0.76 0.06

III 0.94 0.06 0.88 0.06 0.95 0.05

Table 5.6. Hypothesis testing results, showing likelihood ratios (LR),

2. degrees of freedom (df) and probabilities of greater (Pr)

for several variables affecting the probabilities of HP

and RD reactions in Trial 4.

HP RD

Variable df LR Pr(X2 ) LR Pr(X2.

Test 2 49.1 0.000 2.3 0.317

Sex 1 6.2 0.013 0.3 0.584

Test x Sex 2 2.4 0.301 245 0.287

Treatment 2 1.1 0.577 0.4 0.819

Test x Treat 1 1.7 0.192 0.3 0.584

Sex x Treat 2 0.7 0.705 4.5 0.105

Year 1 1.6 0.206 0.6 0.439

Table 5.7. Estimated probabilities (p) of HP reactions for

the different tests in trials 1, 1 & 4, 2 and 3.

Test

Trial Sex I II III IV

1 Males Females

1 and 4 Males (pooled) Females

2 Males Females

3 Males Females

0.41 0.71 0.71 -- 0.41 0.56 0.88 -

0.42 0.72 0.81 -- 0.51 0.73 0.95 -

0.12 0.54 0.47 - 0.27 0.71 0.72 -

0.59 0.84 0.79 0.85 0.69 0.88 0.88 0.95

Table 5.8. Hypothesis testing results, showing likelihood ratios (La), degrees of freedom (df) and probabilities of greaterx(Pr) for several variables affecting the probability of HP reactions in trials 1, 1 & 4 1, 2 and 3.

Variable

Test Sex T x S Years Pigs

LR 36.3 0.1 10.7 122.5 Trial df 2 1 2 -- 64

Pr(7-2 ) 0.000 0.752 0.005 -- 0.000

trials LR 76.5 4.8 6.3 11.3 l and 4 df 2 1 2 2 -- pooled Pr(X) 0.000 0.028 0.043 0.004

LR 212.1 26.1 1.9 -- 484.2 Trial 2 df 2 1 2 - 199

Pr(X 1 ) 0.000 0.000 0.387 -- 0.000

LR 62.9 11.0 2.5 -- --

Trial3 df 3 1 3 -- --

Pr(X 1 ) 0.000 0.001 0.475 --

Page Si

the different tests and sexes in the four trials. As shown in Table 5.10

there were significant effects of individual pigs and years, and even the

test x sex interaction was significant in Trial 1. The frequency of

indeterminate diagnoses , however, was largely unaffected by systematic

sources of variation like test and sex. The exception was Trial 3, where

the differences between tests were significant. The probability of RD

reactions averaged 0.07 over all trials.

HP + RD reactions.

The estimated probabilities of HP+HD reactions for the two sexes at

different ages were very similar, in all trials, to those that can be

obtained by adding the separate estimates for HP and HD, and are therefore

not reported. The conclusions from the statistical analysis followed

closely those obtained for HP reactions.

DISCUSSION

All animals in the present study were . offspring of HP x HP matings;

if a single-locus biallelic model is assumed for mode of inheritance of

susceptibility then, according to the findings in Chapter 3. pigs In

trials 1 and 4 (PTH) will be homozygous recessive and the frequency of HP

will represent the penetrance of that genotype. Animals in trials 2 and

3, however, will be expected to be a mixture of three genotypes since, in

addition to one homozygote, about a half of the heterozygotes were HP in

British Landrace. The expectation for the frequency of HP in trials 2 and

Table 5.9. Estimated probabilities (p) of HD reactions for

the different tests in trials 1, 1 & 4, 2 and 3.

Test

Trial Sex I II III IV

1 Males 0.12 0.03 0.24 - Females 0.25 0.19 0.06 -

1 and 4 Males 0.11 0.05 0.14 - (pooled) Females 0.18 0.11 0.04 -

2 Males 0.08 0.05 0.08 -- Females 0.09 0.06 0.08 -

3 Males 0.07 0.05 - 0.05 0.03 Females 0.08 0.03 0.06 0.02

Table 5.10. Hypothesis testing results, showing likelihood ratios (LR), degrees of freedom (df) and probabilities of greaterx.L(Pr)

for several variables affecting the probability of MD reactions in trials 1, 1 & 4, 2 and 3.

Variable

Test Sex T x S Years Pigs

LR 2.4 0.6 16.0 793 Trial df 2 1 2 -- 64

Pr(X 2 ) 0.301 0.438 0.000 0.094

trials LR 4.5 0.1 11.4 6.7 - l and 4 df 2 1 2 2 pooled Pr(X 1 ) 0.105 0.752 0.003 0.035

LR 2.2 0.4 0.1 - 150.3 Trial 2 df 2 1 2 -- 199

Pr(x 2 ) 0.333 0.527 0.951 -- 0.015

LR 8.0 0.1 1.7 -- --

Trial3 df 3 1 3 -- --

Pr(XZ) 0.046 0.752 0.637

3 will be the sum of the penetrances of the two susceptible genotypes

weighted by their frequencies. The increase in frequency of HP from Trial

2 to Trial 3 could thus be due to selection in favour of susceptibility,

combined with a longer halothane test in Trial 3.

As shown in Figure 5.2, the results from all trials indicate clearly

that penetrance tends to increase between the third and eighth week of

age, both in PTH and in Landrace pigs. This is interpreted as halothane

susceptibility having a variable age of onset (Falconer, 1967). It could

be interesting to investigate whether part of the variation is genetic.

Although the general trend was for penetrance to increase with age, a

proportion of animals exhibited HP reactions in earlier tests but did not

react in later ones; they illustrate another aspect of penetrance. All

pigs that completed 3 tests were classified into eight categories,

according to the sequence of HP reactions they exhibited. For example,

pigs in the category 011 did not react positively in the first test but

did so in the second and third tests. Table 5.11 shows the distribution

of pigs into such categories. Although a variable age of onset may give

rise to systematic changes in penetrance, there is still a proportion of

animals -those in categories 4 to 7- that, having shown susceptibility at

some stage, failed to exhibit HP reactions later on. According to the

results from Trial 4, an effect of previous tests reducing the probability

of HP reactions in subsequent ones is unlikely. Such cases, therefore,

must indicate that random variation in the exertional, nutritional or

health status (Mabry, Christian and Kuhiers, 1981) or even

misclassification can make some susceptible pigs fail to exhibit an HP

reaction, even though they reached their age of onset.

Figure 5.2. Age changes in the probability of HP reactions (p)

estimated in (a) PTH and (b) Landrace.

0 Z0 30 49 DU Di,

Age (days)

-' (a) PTH LO - -

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

(b) Landrace A n of HP

----- /

trial 2

0 20 30 40 so Go

Age (days)

- Females

Males

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

trt

The differences between sexes in incidence of HP were unexpected.

Although a test x sex interaction was detected in PTH (Table 5.8)

penetrance was always higher in females than in males; the effect was

particularly marked in Trial 2 (Figure 5.2). Such differences in

penetrance could not be related to differences in weight or age, as the

sexes were always very similar in those respects; differences in

reaction-time cannot be invoked either (see Table 4.4). The reasons for

this interesting sex effect have still to be investigated.

The finding that penetrance changes with the age of the animals would

have to be considered when studying the mode of inheritance of halothane

susceptibility on pigs of different ages. Batsehelet (1963) has shown how

to incorporate penetrance as a non-decreasing function of age into models

for testing genetic hypothesis and estimating parameters. It could also

be worthwhile to construct such models allowing for penetrance to differ

between sexes.

From a practical point of view, if penetrance is an increasing

function of age an optimum age for testing could be defined, after which

the marginal increments would be considered not worth the while; this is,

naturally, a rather arbitrary concept. Judging crudely from the results

in Landrace (see Figure 5.2) such age could be somewhere around five weeks

of age. Breeds might, however, differ in the functional relationchip

between age and penetrance and so, perhaps, in the optimum age for

testing.

Table 5.11. The distribution of all pigs (%) that received

three halothane tests into categories according

to the sequence of HP reactions they exhibited.

Breed

PTH Landrace

Trial 2 Trial 3 Category* n130 n=198 n=244

001 16.9 10.1 6.1

011 25.4 32.3 20.5

111 37.7 16.2 52.9

4: 100 0.0 0.5 0.8

5: 010 4.6 11.1 6.1

6: 110 3.1 2.5 7.0

7: 101 6.9 0.5 4.1

8: 000 5.4 26.8 2.5

* 0 indicates absence of HP,

I indicates presence of HP.

CHAPTER 6. THE EFFECTS OF HALOTHANE SUSCEPTIBILITY

ON SOME ECONOMICALLY IMPORTANT TRAITS

The incidence of halothane susceptibility varies widely among

different pig breeds; the trait has a relatively simple mode of

inheritance so that the frequency can be changed readily by selection (see

for example Webb, 1981). Therefore, a question of some importance to pig

breeders concerns the breeding policy to adopt in relation to

susceptibility in order to improve production efficiency. At first sight

the occurrence of stress—related deaths among susceptible pigs may call

for elimination. However, there is growing evidence to show that these

pigs exhibit a variety of changes some of which, such as reduced meat

quality, are detrimental while others, such as increased lean content, are

economically desirable (for a review see Webb, 1981). Since a profitable

breeding policy should be based on the economic balance between beneficial

and harmful effects it is necessary to identify all the traits that are

affected and to characterise properly the changes (Smith and Webb, 1981).

A research project was started in 1974 at the Animal Breeding Research

Organisation (ABRO) to investigate several aspects of halothane

susceptibility, including It associat i ons with econic performance. A

Pietrain/Hampshire (PTH) synthetic population, from which susceptible and

tolerant lines have been derived, played an important role in these

investigations. In this chapter the effects of susceptibility on

reproductive, growth and carcass traits are assessed by looking at the

differences between pigs of the susceptible and tolerant ABRO—PTH lines.

It is convenient therefore to begin by analysing the genetic structure of

those lines.

6a THE GENETIC STRUCTURE OF THE ABRO-PTH LINES OF PIGS

(i) Demographic structure of the PTH population.

A series of crosses between Pietrain and HampsHire pigs was started in

1971 at ABRO with the aim of creating a synthetic sire line. Between 1971

and 1974 three batches of matings involving overlapping generations

(designated here as blocks) were carried out. Following a report that

liability to the Porcine Stress Syndrome could be predicted from the

reaction to halothane (Eikelenboom and Ninkema, 1974) the population was

screened for susceptibility and an incidence of about 20 % was found. Two

lines were then formed, by mating mainly reactors with reactors and

non-reactors with non-reactors (these matings provided information for

studies on the genetics of susceptibility: see Smith and Bampton, 1977;

and this thesis). At this stage the population was a mixture of Fl, F2

and F3 pigs and even some backcrosses. The two lines were subsequently

selected for and against positive reactions; they were called the Stress

Susceptible (SS) and Stress Resistant (SR) PTH lines. Only halothane

positive reactors. from litters where all sbs reacted positively, were

kept as replacement breeding stock in SS. All breeding animals in SR were

halothane negative reactors, most from all-negative litters.

In 1973 -one year before the subdivision- four male and three female

immigrants were introduced into the population from a PTH herd at the

University of Newcastle. A year later two new male immigrants were

introduced from Newcastle, this time into the SR line. This immigrant

material played an important role in the genetic make—up of the SR line.

This study covers the first annual batches of matings (blocks) of

the SS and SR lines. Figure 6.1 illustrates the changes in susceptibility

frequency as a result of selection. Tables 6.1 and 6.2 show the

population sizes and the distribution of breeding animals according to sex

and generation. Since some matings. took place between animals of

different generations the convention was adopted of assigning a pig to

generation it + i when its father belonged to generation it.

Two features in Tables 6.1 and 6.2 must be mentioned: neither the

population size nor the age distribution remained constant over the

different blocks. Another interesting feature is that there was more

overlapping of generations in SS than in SR; a reduced conception rate in

SS females' was one of the reasons for such difference (Webb, personal

communication).

(ii) The genetic composition of the SS and SR lines.

The PTH population was founded with 28 animals: 5 Pietrain and 10

Hampshire males, 6 Pietrain and 7 Hampshire females. Nine PTH immigrants

were introduced later into the population as described above. These 37

animals were the source of all genes in the SS and SR lines; they are

designated as founders, A (n = 1,..., 37).In

Figure,6.. Frequencies of halothane positive reactions in the

SS and SR ABRO-PTH lines.

1.00

0.75

I ::: 0.00

I) I Z i

Block

Table 6.1 Population size and distribution of the breeding animals

according to sex and generation in the SS line.

Males

Generation Total

Block 1 , 2 3 4

1 6 6

2 - 3 3

3 - 7 4 11

4 -- 2 2 7 11

5 -- 1 1 6 4 12

Females

Generation Total Total

1 2 3 4 5 population

25 25 31

- 6 6 9

-- 11 7 18 29

-- 5 3 4 12 23

-- 4 1 9 11 25 37

Table 6.2. Population size and distribution of the breeding animals

according to sex and generation in the SR line.

Males Females

Generation Total Generation Total Total

Block 1 2 3 4 5 d' 1 2 3 4 5 population

1 10 10 27 27 37

2 -- 5 5 —14 14 19

3 - 3 4 7 --1110 21 28

4 ---- 2 9 11 -- 1 423 28 39

5 ------- 8 8 ---------2929 37

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For any individual P in SS or SR there is a vector of values

. #i , representing the probabilities that a gene,

131

taken at random from P. , is a copy of a gene in founder A . The vector of

mean values of over all members of block i (=1,..., 5) in line k (SS

or SR):

= [~ • jK,4

characterises the line, at each stage, in terms of origin of genes

(Jaquard, 1974).

For each individual P. the probabilities Øi were calculated from the

occurrences of A in the pedigree. For example, if A was parent of P

then 09 = 1/2; if it was a grandparent then ø9 = 1/4 and so on; if

A occurred more than once in a genealogy the probabilities from each line TI

of descent were summed. A computer program was written to derive the

probabilities . from the pedigrees.

Tables 6.3 and 6.4 suinmarise the results of these calculations. In

these tables the founders were divided into three groups: the 10 Pietrain

or Hampshire pigs which contributed most to the genetic make-up of the

lines are. shown singly and as a group; the contributions from the

immigrants and from the remaining founders are given as group

contributions only. The tables also show the expected proportions of

Pietrain and Hampshire genes, calculated by assuming the genetic

composition of the 9 immigrants to be 50 % Pietrain and 50 Z Hampshire

(Webb, personal communication).

Table 6.3. Probabilities of origin of genes from the different

founders in the SS line.

Block Breed of founder Founder 1 2 3 4 5

10 0.028 0.049 0.047 0.048 0.051 80 0.048 0.042 0.037 0.036 0.033

Pietrain 6826 0.085 0.083 0.074 0.073 0.076 7567 0.032 0.028 0.039 0.046 0.039 8054 0.065 0.097 0.080 0.077 0.086

89 0.065 0.097 0.080 0.077 0.086 5493 0.089 0.070 0.058 0.068 0.066

Hampshire 1035 0.089 0.090 0.083 0.089 0.092 1353 0.137 0.125 0.125 0.146 0.138 2793 0.028 0.049 0.047 0.048 0.051

10 principal founders 0.666 0.730 0.670 0.708 0.718

Other 18 founders 0.318 0.270 0.278 0.292 0.270

Immigrants 0.016 0.000 0.052 0.000 0.012

Total 1.000 1.000 1.000 1.000 1.000

Pietrain 0.390 0.390 0.405 0.384 0.385

Hampshire 0.610 0.610 0.595 0.616 0.615

Table 6.4. Probabilities of origin of genes from the different

founders in the SR line.

Block Breed of founder Founder 1 2 3 4 5

10 0.061 0.020 0.036 0.036 0.032 80 0.051 0.043 0.047 0.046 0.040 6826 0.044 0.049 0.054 0.044 0.039 7567 0.027 0.033 0.029 0.021 0.016 8054 0.034 0.026 0.029 0.038 0.033

89 0.034 0.026 0.029 0.038 0.033 5493 0.057 0.066 0.063 0.054 0.047 1035 0.065 0.049 0.051 0.045 0.039 1353 0.122 0.109 0.109 0.094 0.078 2793 0.061 0.020 0.036 0.036 0.032

10 principal founders 0.556 0.441 0.483 0.452 0.389

Other 18 founders 0.304 0.192 0.212 0.222 0.204

Immigrants 0.140 0.367 0.305 0.326 0.407

Total 1.000 1.000 1.000 1.000 1.000

Pietrain 0.386 0.424 0.420 0.426 0.438

Hampshire 0.614 0.576 0.580 0.574 0.562

Pietrain

Hampshire

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The results of the analysis indicated that the different founders

contributed rather unevenly. Many of them were scarcely represented in

the composition of the lines; this must have produced some loss of

genetic variance. The results also indicate that the contributions of

some Pietrain and Hampshire founders differed between the lines, as in the

case of male Hampshire 89 or female Hampshire 1353. The most important

finding, however, was the differential contribution of the immigrants to

the composition of the lines. In numbers they represented about 24 % of

the founder group. However, while they contributed some 40 % of the genes

to the last block of SR, the corresponding contribution to SS was almost

nil. The composition of the two lines in terms of Pietrain and Hampshire

genes was quite similar: about 40 and 60 % respectively.

(iii) Random genetic differentiation of the lines.

As inferences about the effects of halothane susceptibility on several

quantitative traits are to be based on differences between pigs of the SS

and SR lines it is important to estimate the expected random

differentiation of the lines, due to genetic sampling and immigration.

Any estimate of the drift variance based on an effective population number

(N ), as in Hill (1972), is bound to be very imprecise because there are

no formulae for N€ capable of accommodating the complicated population

structures shown in Tables 6.1 and 6.2 or of taking into account the

differential immigration. The genealogical information, however, offers a

way round these difficulties. The following derivation was suggested by

Dr. W. G. Hill (personal communication). Consider a quantitative trait

controlled by a single locus with two alleles and write p and (1 - p) for

the allelic frequencies in a large, random mating population. In the

absence of dominance the mean of this population will be

a (2p - 1)

where a is the difference between homozygotes and heterozygotes (e.g.

Falconer, 1981; pp 101). Let a pair of lines, j and k, be derived from

this base population. The variance of differences in means between such

pairs of lines is

V(,Zz. V[ (a ( 2 -1)) - (a 1))]

= 4a2 V(p. - p ) ....... ..............(1)

The variance of the difference in allelic frequencies is

- p ) = E(p? ) + E(p 1 ) - 2E(p p. )

with

E(p ) p + (1 -/3 , )P 2•

where is the mean coancestry coefficient among members of line J.

Similarly

2. E(p 'K

p + (1-113. )p

where is the mean coancestry of members of j with members of k.

Therefore

V(p3 - p ) p(l - )(/ /3K

= p(l - p) DJ ................. (2)

The quantity

= /J

D Jit . i3.+/2 -2/3.

/ k /jK

is called here the random genetic differentiation between lines j and

k. Replacing (2) in (1)

V( p. _,(LK) = 4p(l - p) a DJ ft

= 2 crA D *K ................ ( 3 )

where T represents the additive genetic variance (e.g. Falconer,

1981; pp 116); the same formula applies for polygenic traits determined

only by additive genes and is an approximation otherwise.

The mean coancestries for each block of the SS and SR lines ( /3 and / Ss

) were estimated by taking a sample of animals and calculating the SR

coancestries among all possible pairs, including animals with themselves.

Twenty pigs were sampled from each block/line, except when population size

was less than 20, in which case the coancestries were calculated amongst

all animals in the block. When numbers permitted, 10 males and 10 females

were sampled. The mean coancestries among members of SS with members of

SR (1S ) were estimated on the same samples that were used for / SS,SIt

estimating the within—line coancestries. The block previous to the

separation of the lines was taken as the base population. All

computations were carried out with a computer program that calculates

coancestries and inbreeding coefficients (W. G. Hill, personal

communication).

The evolution of the coancestries within and between lines together

with the random genetic differentiation during the period covered

by the study, are shown in Table 6.5 and in Figure 6.2.

There is a potential bias in taking equation (3) as the expected

Table 6.5. Changes in the estimated mean coancestry coefficients

( 1 3 ) within and between lines, and in the random

genetic differentiation between SS and SR (Dss sm

Mean coancestry

Between Within lines lines

Block /'sp SS, $a

1 0.0339 0.0224 0.0259 0.0045

2 0.0209 0.0401 0.0253 0.0104

3 0.0499 0.0604 0.0204 0.0695

4 0.0652 0.0742. 0.0250 0.0894

5 0.0835 0.0987 0.0178 0.1466

Figure 6.2. The evolution of the average coancestry (jS) within

the SS and SR lines, and of the random genetic

differentiation ) between the two lines.

0.14 0 '.4

0.12

Old w 0.10 4 1

lj 1.4 1J ,-I

0.08 ('U !I

:::

0.02

tlsssR

)SR

I3 s

0 1 2 3 4 5

Block

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drift' variance of. the difference between populations SS and SR in

quantitative traits. The bias arises because the two lines were brought

apart by selection for and against halothane susceptibility. In the

present case the variance of the random genetic differentiation should be

2 2.

- ) 2 -1 )D A AS SS,S

approximately. The term .a_ represents the additive varianceAS

controlled directly by the locus (or loci) determining susceptibility.

Therefore, for traits affected by the halothane gene equation (3) yields

overestimates of the true random differentiation variance. It is not

possible to make allowances for such source of bias in this study;

nevertheless, when considering the opportunities for random changes

between SS and SR because of small population size, genetic - bottlenecks'

and differential immigration it seems only desirable to have some

approximate, albeit biased, idea of the size of the drift variance.

6b DIFFERENCES IN REPRODUCTIVE TRAITS BETWEEN HALOTHANE

SUSCEPTIBLE AND HALOTHANE TOLERANT PIGS

Of all the changes in economic traits that are presumably brought

about by halothane susceptibility those concerning reproduction are

perhaps among the less well documented. In his comprehensive review of

the literature Webb (1981) quoted only two studies (Webb and Jordan, 1978;

Schneider, Schwrer and Blum, 1980) as reporting halothane susceptibility

effects on reproduction. While the results of both studies agree in that

susceptible females have a reduced litter productivity such effects are

far from being firmly established. In general, reliable estimates of

reproductive performance are required to optimise the use of genetic

resources available to pig production, particularly with crossbreeding

(Smith, 1964; Moav, 1966); the requirement still holds when a breeding

strategy has to be decided with halothane susceptibility (Smith, 1981).

The objective of this study was to assess the effects of halothane

susceptibility on some litter traits by looking at the differences between

reacting and non-reacting - females from the SS and SR ABRO-PTH lines of

pigs.


Animals.

Two data sets were available. The first one comprised 206 litter

records from females in blocks 1 to 5 of the SS and SR lines. All females

were mated to boars from their own lines. The farrowings took place in

winter; concrete floored farrowing pens were used. The second data set

consisted of 93 records of females from blocks 3, 4 and 5 of the SS and SR

lines which, after the winter farrowing, were re-mated to boars from the

SR line. The purpose of such matings was to produce animals for the

carrier trials. Briefly, if susceptibility Is a reccsIvc trait

controlled by a single locus, pigs from SS are expected to be homozygous

recessive (nn); most pigs from SR are expected to be homozygous normal

(MN). Offspring from SS dams with SR sires are expected to be mostly

heterozygotes (Nn); SR x SR matings are expected to produce mainly NN

progeny. The objective of the carrier trials was to compare NM versus

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Mn pigs for several growth and carcass traits. A fuller description of

these trials will be given later. These farrowings took place in summer;

field arks were used as farrowing pens. The piglets were weaned, on

average, at 50 days of age.

Six traits were studied, which were treated as traits of the mother:

(a) At birth:

Litter size = (live + stillborn),

Average piglet weight = (litter weight)/(number of piglets),

Perinatal mortality = number of stillborn piglets.

(b) At weaning:

Litter size,

Average piglet weight,

Mortality during lactation = deaths from birth to weaning.


I. Analysis of litter size and piglet weight.

Differences between the SS and SR lines in litter size and in mean

piglet weight, at birth and at weaning, were estimated by the method of

least-squares after fitting different linear models to the data with the

computer program LSML76 (Harvey, 1977). Three separate analyses of

variance were done:

(1) Within—line matigs: all records. The following linear model was

used

Y = ,M + B. + + P )( + (BL).. + (BP). + (LP). + e.. A,) J

where Y<e is the size (or mean piglet weight) of the lth litter, of

kth parity (1, 2 or 3 and higher) in the jth line (SS or SR) and in the

ith block (1,..., 5). Some females had multiple records, the analysis

however assumed that all records came from different females. When piglet

weights were studied litter size was included as a covariate in the model.

Within—line matings: first parities. The previous analysis

ignores the family structure of the population and probably underestimates

line—mean variances. Therefore, it may yield unreliable tests of

hypotheses. However, it was difficult to fit a model accounting for

family relationships since a complex population structure produced several

unconnected groups of data. Therefore, the subset of all first parities

(165 records) was re—analysed, using the following hierarchical model:

.. Y. =)+ (BL) +SAj AJK + -L.Kt +e

where Y.. is the performance of female m, the daughter of dam I and .4-i <tn

sire k, within line j (SS or SR) and block 1 (1,..., 5). A small number

of sires and dams were counted twice because they appeared in two z

different blocks. This analysis allowed the estimation of the sireIs ;

variance component.

Carrier trials. The statistical model fitted to the 93 records

from this data set was the same as that in (i); the effect of the ith

block was here for i = 3, 4 or 5. No attempt was made to account for the

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family structure of the population because the data set was very small.

The analysis was intended as a tentative examination but is reported for

completeness.

Table 6.6 summarises the degrees of freedom and expected mean squares

for the three analyses of variance.

(iv) Variance of line differences. For each trait the variance of the

difference between SS and SR was assumed to be

2. '2. V(SS— SR) = ;

2. represents the error variance of the line difference, arising from

2. estimating genetic means from phenotypic means; is the variance due

to random genetic differentiation (Hill, 1981).

The error variance was estimated as

7- - _z e(SSSR) IF

where F is the appropriate variance ratio from the analysis of

variance. The drift variance was estimated from equation (3) as

2. =20 D

d n SS 1SR

2 2 where tT. , the additive genetic variance was taken as 4 T and

was the mean value of (Table 6.5) over the i blocks

considered in the analysis.

Table 6.6. Degrees of freedom from the analyses of variance

of litter sizes and piglet weights in the within-

line matings and in the 'carrier trials.

Within-line matings

All First Carrier Source records parities trials

Block (B) 4 - 2

Line (L) 1 - I

Parity (P) 2. - 2

B x L 4 9 2

B x P 5 - 3

L x P 2 - 2

Sires/(B x - 55 -

Dams/Sires/(B x - 33 -

Remainder 187 67 80

2.

* Expected value of dam mean square: + 1.66 , and of sires

2 21 2 2. 1 2

mean square: 11., + 1.64ç + 2.38 T°'D and 1s are the

within-litter, dams and sires variance components respectively.

The null hypothesis that differences between SS and SR were different

from zero was tested by means of a t-test.

II. Analysis of piglet mortality.

The probabilities of perinatal deaths and of deaths during lactation

in litters of SS and SR sows were estimated by the method of maximum

likelihood. The procedure was similar to that used in Chapter 5 when

studying age effects on halothane susceptibility. The number of deaths in

the ith litter, dL (1 = 1,..., ), was considered to be a binomial random

variable. The total likelihood was

L = rt

.=1

where n. is the number of piglets in the ith litter. The

probabilities of deaths, p. , were linked to linear functions of several

explanatory variables by means of the logit transformation (Finney, 1970).

Maximum likelihood estimates of the parameters in the linear functions

(MLE) were obtained after maximising log L with respect to those

parameters; this was done with the computer program GLIM (Baker and

Nelder, 1978).

Analyses using models (i), (ii) and (iii) were repeated for the two

mortality traits; the same linear functions were used, but including now

the number of piglets born as a covariate. The statistical significance

of the line effects was tested by means of the likelihood ratio

criterion (LR, as in Chapter 5), after fitting linear models with and

without the L set of parameters; the LR was compared with a

distribution. No attempt was made to estimate the drift variance (Od

in these traits.

RESULTS

Tables 6.7 and 6.8 show the estimated differences between SS and SR

females in litter and mortality traits. In general terms the estimates in

Tables 6.7 and 6.8 show SS females as producing about 1.5 piglets

less than the SR, probably as a result of smaller litters at birth and

higher lactation mortality. There were no indications of differences in

piglet weights. Most of the differences were estimated with low precision

and only a few reached statistical significance.

DISCUSSION

The small sizes of the SS and SR populations proved to be an important

source of error in the estimation of changes associated with halothane

susceptibility; in most cases the drift variance more than doubled the

error variance. In spite of this difficulty the differences in litter

size at weaning were found to be significantly different from zero in a

consistent way. The question may be asked whether such differences were

caused by differential mortality during lactation or by both differences

in prolificacy and in mortality. This might concern pig farmers since it

could be more feasible for them to reduce lactation mortality by

management than to alter prolificacy differences. Unfortunately there is

Table 6.7. Overall means, standard deviations (SD) and estimated differences

between females of the SS and SR lines for some litter traits, in

the within-line matings (WL) and in the carrier-trials.

Difference Overall

Trait mean SD SS - SR SE SE b

No. records

WL, all records 86 vs 120 WL, first parities 56 vs 109 Carrier-trials 36 vs 57

Litter size at birth (piglets)

WI., all records 8.6 2.91 -1.07 0.438 0.873 WI., first parities 8.3 2.85 -1.01 0.577 0.951 Carrier-trials 10.1 2.86 -1.27 0.470 1.063

Piglet weight at birth (kg)

WL, all records 1.2 0.24 -0.07 0.036 0.048 WI., first parities 1.2 0.24 -0.06 0.048 0.055 Carrier-trials 1.3 0.20 -0.12 0.063 0.077

Litter size at weaning (piglets)

WI., all records 6.0 3.29 -1.56 0.498 0.804 * WL, first parities 5.8 3.42 -1.84 0.637 0.897 * Carrier-trials 7.5 2.82 -1.88 0.379 0.881 *

Piglet weight at weaning (Kg)

WI., all records 11.0 5.26 -0.67 0.624 1.050 WI., first parities 10.3 5.17 -0.76 0.646 1.064 Carrier-trials 13.6 4.24 0.50 1.151 1.568

* P < 0.05 after a two-tailed t-test.

standard error, not including the drift variance.

standard error, including the drift variance.

Table 6.8. Maximum likelihood estimates (MLE) of the probabilities of

deaths (7.) in the within-line matings (WL) and in the

carrier-trials.

MLE

Trait SS SR Difference 1/

Perinatal mortality (%)

WL, all records 8.60 7.40 1.20 NS

WL, first parities 13.90 10.58 3.32 NS

Carrier trials 7.76 5.65 2.11 NS

Lactation mortality (%)

WL, all records 33.33 13.36 19.97 ***

WL, first parities 32.73 28.71 4.02 ***

Carrier-trials 29.20 15.99 13.21 ***

1/ Random genetic differentiation not taken into account when

testing these hypotheses.

P < 0.001 after the LR was compared with a X distribution.

not a clear-cut answer: on the one hand the differences in lactation

mortality were large, but there are doubts about the role played by drift

in the differentiation of the lines. On the other hand, although the

differences in litter size at birth were not significant they were large

and consistent with the results at weaning, and it could well be that the

analysis simply lacked the power to detect such differences. Thus, while

providing indications that susceptible sows had smaller litters at weaning

because of both reduced prolificacy and increased lactation mortality this

study falls short of proving that this interpretation is correct.

Schneider et al. (1980) also found that halothane susceptible sows were

less prolific than halothane resistant sows.

The fact that there are differences between the SS and the SR lines in

any given trait does not neccesarily mean that they are pleiotropic

effects of the halothane susceptibility gene. The reason is that the PTH

population consisted of a mixture of Fl, F2 and F3 Pietrain x Hampshire 0

crossbred pigs when the two lines were founded. Therefore, Pietrain and

Hampshire genes were not expected to be randomly associated at that stage.

If, as suggested by Smith and Bampton (1977), the susceptibility genes

came into PTH mainly from the Pietrain breed, the first cycle of selection

would have made them 'hitch-hike' a piece of Pietrain chromosome of

variable length into the SS. line. If any locus affecting the trait in

question happened to be in these pieces of chromosome, and if the Pietrain

and Hampshire populations differed in gene frequencies at this locus, a

difference would have arisen between SS and SR due to the hitch-hiking

effect.

This possibility is particularly relevant in the case of litter size.

It has been found that H, a red-cell antigen locus cioseiy linked to the

halothane locus (Andresen and Jensen, 1977), has effects on reproductive

traits in pigs (Jensen et al., 1968; Rasmusen and Hagen, 1973). Both

studies agree that the H a

allele would reduce litter size. It is

Important to emphasise that these findings were made in breeds where

halothane susceptibility is now virtually absent, such as the Duroc (Webb,

1981). Therefore, it is possible that gene substitutions at loci linked

to the halothane locus have an effect of their own on litter size.

Imlah and Thompson (1979) surveyed the allelic frequencies at the H

locus in the founder PTH group and in the first block of the SS and SR

lines. They found that halothane susceptibility was associated with a.

and that there was a large excess of this allele in SS. Therefore, there

is a distinct possibility that a gene with effects on litter size might

have been hitch-hiked Into SS by the halothane gene, due to linkage

disequilibrium in the founder group. If this was the case, at least part

of the differences between SS and SR would be due to gene frequency

differences at loci other than the halothane locus. Obviously, this

would Imply that the present findings need not apply to all pig

populations. Also, that it might be possible to dissociate halothane

susceptibility from the litter size effects in SS by selecting for

appropriate recombinants. It would be Interesting to assess the present

gene frequencies at the H locus In the SS and SR lines.

The finding that postnatal mortality in litters of halothane

susceptible sows was very similar both in the within-line matings and in

the carrier trials is somewhat surprising. Although all traits were

treated as traits of the mother this working hypothesis was assumed in

order to set up the analysis. In fact, it was expected that the genotype

of the piglets would have been an important source of variation,

particularly for lactation deaths, after Eikelenboom et al. (1978) found

higher postweaning mortality among susceptible pigs when compared with

normal pigs. Under the single-recessive hypothesis the progeny from SS

sows in the within-line matings were expected to be halothane susceptible

(nn) while those in the carrier trials were expected to be normal (Nn).

The fact that the incidence of deaths was very similar in both groups,

while there were important differences between halothane susceptible and

normal sows, suggests that mortality during lactation is largely a

maternal trait in PTH.

The differences shown in Tables 6.7 and 6.8 are large enough to have

practical implications for pig farmers. However, since they were

estimated with low precision, and as the possibility exists that they are

peculiar to the ABRO-PTH population, it would be desirable to verify the

present findings in commercial pig populations.

6c DIFFERENCES BETWEEN PIGS OF PREDICTED GENOTYPES AT THE HALOTHANE

LOCUS IN GROWTH AND CARCASS TRAITS

In recent years there has been considerable research on the effects of

halothane susceptibility on production traits in pigs. The results

consistently show that reactors are leaner and have higher carcass yields

than non-reactors. Such desirable effects are opposed, on economic

grounds, by lowered meat quality, reduced female productivity and

increased postweaning mortality (for a review see Webb, 1981). If the

question arises today as to the consequences of selecting for or against

susceptibility there will be information available on which to base an

economic balance. Elimination or fixation, however, does not exhaust all

breeding alternatives. The advantages in carcass traits might be

exploited by using specialised sire and dam lines; the adverse effects on

reproduction might be confined to susceptible sire lines which could be

crossed with non-susceptible dams to produce commercial offspring, as

first suggested by Minkema, Eikelenboom and van Eldik (1976). The

economic analysis of such a strategy demands knowledge of the mode of

inheritance of suseptibility and of -the genotypic values for all

production traits (Smith and Webb, 1981). The same information is

required for predicting what correlated changes in susceptibility

incidence are to be expected given any selection program for economic

efficiency. Concerning genotypic values, though, the information in the

literature is still rather sparse (see Webb, 1981) and more research seems

to be required.

After it was considered that a single-recessive model represented

satisfactorily the mode of inheritance of susceptibility in the PTH

population (Smith and Bampton, 1977) a series of trials was started at

ABRO in order to estimate genotypic values for several traits of economic

relevance. The objective of these trials was to estimate the differences

between the heterozygous (Nn) and the homozygous normal (NN) genotypes.

The possibility of estimating all three genotypic values at the halothane

locus was foregone in favour of this particular comparison. Given a

fixed amount of resources it was preferred to estimate with better

precision the differences between normal and carrier pigs, on which the

economic benefit of exploiting specialised sire and dam lines would mainly

depend. Seven trials were carried out at ABRO for this purpose, based on

three different experimental designs (Webb, 1981). In this section the

analysis of three such trials based on PTH pigs is presented. Some

preliminary results have been reported earlier by Webb (1981).


Animals.

Two groups of pigs were performance tested in three trials. The two

groups were putative heterozygotes (Nn) vs putative homozygous normals

(NN). They were offspring of dams from the SS and SR lInes respectively

mated to sires of the SR line. The experimental design was thus a

within-sire comparison of Nn vs NN pigs. The three trials corresponded to

the third, fourth and fifth blocks of matings in the PTH lines. The

desired genotypic composition for the progeny was:

SIRE DAM PROGENY.

(SS) nn Nn

(SR) _______

(SR) NN NN

All animals in the present series of trials were given one halothane

test at about eight weeks of age, following Webb and Jordan's (1978)

procedure; only negative reactors were included in Nu; the NN group

comprised only negative reactors from all-negative litters. Since sires

and dams were not progeny tested prior to the trials, however, the

possibility that the NN group included a small proportion of heterozygotes

(Nn) cannot be ruled out.

Performance testing.

The pigs were weaned at an average age of fifty days. Two full-sib

pairs from each litter were then sent to the Meat and Livestock (MLC)

testing station at Stirling. On some occasions only one full-sib pair per

litter was tested. Most pairs consisted of a castrated male and a gilt

although there were some hog:hog and gilt:gilt pairs as well. Each pair

was allocated to a pen indoors and fed twice daily to appetite a standard

ration containing 13.2 NJ/kg digestible energy and 16.8 % crude protein.

The test started when the pair reached 54 kg total weight and was

completed when it reached 165 kg total weight. Both pigs were then

rage LiD

slaughtered. For details of the testing procedure see Buck (1961). The

following traits are studied here:

I. Growth traits. Measured on a pen basis for each pair.

Age at start of test.

Daily weight gain on test.

Daily food consumption.

Food conversion ratio (food/gain).

II. Carcass traits. Measured on all pigs.

Back-fat thickness: average of the shoulder, mid-back and loin fat

thickness, as defined by Smith, King and Gilbert (1962).

Side-fat thickness: average of theC and K depths of subcutaneous

fat, following Smith et al. (1962).

Eye-muscle area: the area of the muscle longissimus dorsi, as

defined by Smith et al. (1962).

Carcass length: measured from the anterior edge of the symphisis

pubis to the anterior edge of the first rib.

Carcass yield: the cold carcass weight as a percentage of the last

live weight.

Trimming percentage: the weight of the trimmed carcass (ex kidney,

fat, kidneys, psoas muscles, feet and head) as a percentage of the

cold carcass weight.

Hind-quarters percentage: the weight of the ham, rump and

rumpstreak joints (see Figure 6.3) as a percentage of the cold

carcass weight.

Carcass pH: recorded on the m. longissimus dorsi 90 mm

post-mortem.

Neat colour: light reflectance as measured with an Evans

Electrical Ltd (EEL) reflectometer on the surface of the muscle

longissimus dorsi.

III. Rumpback traits. Dissections of the rumpback joint (see Figure

6.3) were carried out for one randomly chosen pig in a sample of pens,

according to the method described by Cuthbertson (1968). The following

traits are studied here:

Weight of rumpback joint.

Lean percentage.

RU

RIB f.

Page 118

Figure 6.3. Standardized joints used in )fl..Cs

pig dissection technique.

Fat percentage.

Bone percentage.

IV. Full dissection traits. The left hand sides of the carcasses in

a sub-sample of the rump-back dissected animals were divided into six

joints (see Figure 6.3) which were defined by reference to skeletal points

(Cuthbertson, 1968). Each joint was then dissected into lean,

subcutaneous fat, intermuscular fat, bone and remainder. The following

traits are studied here:

Lean percentage: 200 x (weight of m.psoas + lean in left side)/

carcass weight.

Fat percentage: 200 x (weight of dissected fat in left side)/

carcass weight.

Bone percentage: 200 x (weight of bone in left side) / carcass

weight.

The weights of lean, dissected fat and bone in each of the six

joints.


(1) Analysis of growth, carcass and rumpback traits.

These traits were analysed on a pen mean basis; In total, information

from 126 pens was available. The information on growth traits (I) was

available as pen means. For the carcass traits (II) the pen means were

calculated from the individual measurements. For the rumpback traits

(III) the pen means were estimated by the method of subsainpling with

regression of Conniffe and Moran (1972). From each pen i one observation

was available, from pig 1 say, on each rumpback trait: R 41J (j =

Observations on carcass traits were available on both pigs: and

where k =1,..., 9 denotes the kth carcass trait in (II) above. Each

pen mean for rumpback trait j was estimated by

A 9 R. R, +L J3 .(C —c )

U IL3 z KJ 2iI. 4iK K= 1

where the J1C

are estimates of the partial regression coefficients of

rtunpback trait R7 on carcass trait CK. The coefficients werewere obtained

from a multiple regression analysis using the following model

R M+ g S +Se

m +P

ij . +(SxP) +

Tnnj

E (C ) +^ (carcass weight) + e OK /. 40 innoj

where R - is the value of the fth rt'mpback trait in the oth pig, a

offspring of siren , of the mth sex (hog or gilt) and the nth parity (1 or

2); COK Is the value of the kth carcass trait in the oth pig. This

model was fitted to the data from the rumpback dissected pigs. The

residual variance from these analyses was called the prediction variance'

Pr

2 (0).

The pen means were then subjected to a least-squares analysis of

variance. The model used was

Y .. = LL + T. + S.-. + C + (TG). + (SG).. 1< + I.. .L}<

+P +Se +e

where Y.. is the mean of -the oth pen, a full-sib pair of the nth 4J K eTn•no

sex (Se = hog:hog, hog:gilt or gilt:gilt pair types) born in the mth

parity (P 1 or 2 and subsequent) of the tth dam, within the jth sire,

within the ith trial CT = 1, 2 or 3) belonging to the kth genotypic group

(C = Nn or NN). The following partial regressions were added to the above

basic model: initial weight, for the analysis of growth traits; carcass

weight, for the analyses of carcass and rumpback traits; carcass

temperature, for the analysis of muscular pH. The sire variance component

( f ) was estimated in these analyses. All models were fitted to the data

by using the computer program LSML76 (Harvey, 1977); Table 6.9 shows the

degrees of freedom from the analyses of variance; the significance of the

genotypic effects was tested against the mean square of the sire x

genotype (SC) interaction.

(ii) Analysis of the full dissection results.

Information from 36 fully dissected carcasses from trials 2 and 3 was

available. Eighteen carcasses from each genotypic group had been

dissected. The pigs (all castrated males) were offspring from 15 sires:

7 sires were represented by two progeny each (1 in each genotypic group);

Table 6.9. Degrees of freedom from the multiple regression analysis of

rumpback traits on carcass traits and from the analyses

variance of pen means.

Pen-means analyses

Multiple Growth and Rumpback Source regression carcass traitsa traits 6

Trials (T) - 2 2

Sires/Trials (S) 19 19 19

Genotypes (G) - 1 1

T x G - 2 2

S x G - 15 15

Dams/Sires (D) - 68 66

Parity (P) 1 1 1

Sex (Se) 1 2 2

PxSe 1 - -

Regressions 10 1 1

Remainder 129 89 67

Expected value of sire mean-squares:

2 2 a: + 2.27 + 6.88 2

+ 1.99 + 5.72

1 2 2 where , t1, and TW are the sire, dam and

1/2 within litter variance components respectively.

Page lZi

5 sires had four progeny each (2 in each genotypic group); finally, 2

sires had only one progeny each.

Three least-squares analyses of variance were carried out. In the

first, - the differences between genotypes in percentage of tissue t (lean,

fat or bone) in the carcass were estimated after fitting the model

Y.. =, + S. + G.. (carcass weight) LJKt 4.t it

(carcass weight) +

where Y.. represents the percentage of tissue t in the carcass of A.JI(t

the kth pig, an offspring of the ith sire (1,..., 15) in the jth genotypic

group (Nn or NN); 113 3t is the deviation of the regression coefficient

within genotype j from the common regression coefficient/k

The second analysis was intended to quantify the relationship between

carcass and rumpback % tissue compositions. The following model was

fitted to the full dissection results

,M. + S •+ G,j +/S4 (carcass weight) +

+ (Z of t in ruxnpback) +,. (% of t in rumpback)

+ e &j...

Kt -

with terms as in the previous model.

In a third analysis the following model was used to estimate genotypic

differences in tissue weight distribution:

Z =,u+ S. +G. + (t in carcass)

+/3. (t in carcass) + e..te

where Z.. is the weight of tissue t (lean, fat or bone) in joint I

(hand, collar, ribback, streak, ham or rump) of pig k, an offspring of

sire i (1,..., 15) in genotypic group j (Nn or NN);/jt is the deviation

of the regression coefficient within group j from the common regression

coefficient

(iii) Variance of genotypic differences.

The variance of the difference between Nn and NN was assumed to be

2 1 - ) = + /2CL e.

2 where cr is the error variance; the drift variance ( ) was

halved here because only the dams came from different lines; all sires

came from the SR line. The error variance was estimated as

2 (Nn - NN) /F

where F is the appropriate mean square ratio from the analysis of

variance. For the rumpback traits the error variance was multiplied by

the ratio

( ci; + 1/2 T pr / CW

2 in order to account for prediction errors; T. is the error mean

square from the pen mean analysis and TP, is the 'prediction variance'

(R. A. Sutherland, personal communication). The 'drift' variance was

estimated by

2 2. = 2 Ta D s1sR (5)

2. 2.

where ci , the additive genetic variance, was taken as 4

D SS,SB. (5) is the random genetic differentiation between the SS and SR

lines in the fifth 'block' of matings (Table 6.5). It was not possible to

estimate the 'drift' variance for the full dissection traits as the data

2. set was too small to allow estimation of T . The statistical tests in

(ii) are thus expected to overestimate the significance of the genotypic

differences.

RESULTS

Table 6.10 shows the overall means, standard deviations and the

estimated differences between genotypes in growth, carcass and rumpback

traits. All the differences were estimated with low precision and only

those in meat colour reached statistical significance (Pv 0.05); meat was

paler amongst heterozygotes. Barring the lack of precision, there were

indications that the heterozygotes were leaner than the homozygotes. The

subcutaneous fat depths were consistent with the differences in the

rumpback joint: at a constant carcass weight the rumpback was apparently

Table 6.10. Overall means, standard deviations (SD) and estimated differences

between Nn and NN ABRO-PTH pigs in performance test traits.

Difference Overall

Trait mean SD Nn - NN SE SE b

Growth traits.

no. pens 54 vs 108

Age at start (days) 88.3 9.22 1.0 1.84 2.79 Daily weight gain (g) 662.1 56.81 13.0 9.30 17.47 Daily food consumption (g) 2000.1 124.23 25.0 24.30 33.75 Food conversion ratio 3.03 0.197 -0.02 0.024 0.070

Carcass traits.

no. pens 54 vs 108

Carcass weight (kg) 65.6 2.18 Backfat thickness (mm) 28.6 3.44 -1.3 0.66 1.05 Sidefat thickness (mm) 22.9 3.69 -1.1 0.84 1.17 Eye muscle area (cm ) 32.3 2.46 0.4 0.29 0.57 Carcass length (mm) 720.0 17.08 -1.8 2.81 6.88 Carcass yield (7.) 78.1 1.38 0.3 0.17 0.37 Trimming (7.) 84.9 0.56 0.1 0.11 0.18 Hindquarter (7.) 46.7 0.98 -0.1 0.15 0.31 pH 90 6.1 0.76 0.0 0.06 0.09 Meat colour (EEL) 43.1 3.53 1.6 0.75 0.90 *

Rumpback traits.

no. pens 54 vs 84

Carcass weight 65.7 2.23 Rumpback weight (g) 3893.8 269.36 -72.0 65.08 83.47 Lean (7.) 52.2 3.67 1.5 1.05 1.36 Fat (7.) 30.9 4.34 -1.9 1.22 1.58 Bone (7.) 10.7 1.69 0.2 0.56 0.69

* ! < 0.05

standard error, not including the drift variance.

standard error, including the drift variance.

leaner in Nn pigs. Such results, in turn, agreed with those from the full

dissections. There was a close association between % lean and fat in the

carcass with those in the rumpback joint (Table 6.11). Thus, there were

clear indications that Nn pigs could have about 1.5 Z more lean in the

carcass than NN pigs in the ABRO-PTH lines. All the differences in growth

traits were small and their standard errors comparatively large.

The full dissections also provided suggestions of differences in

tissue weight distribution. At a constant lean weight Nn pigs apparently

had less lean in the ribback and in the streak and more in the rumpback

(Table 6.12). Similarly, at a constant fat weight Nn animals had more fat

in the rumpback (Table 6.13). Finally, there were indications of

genotypic differences in the rate of bone growth in some joints relative

to total bone growth (Table 6.14); such a difference was not observed in

the lean and fat tissues.

Contrary to what was found in the pen-mean analysis (Table 6.10), the

genotypic difference in rumpback weight was statistically significant in

the sub-sample of fully dissected pigs (Nn = 4036, vs NN = 3904 g). The

fact that the former group included hog:hog, hog:gilt and gilt:gilt pairs

while the latter only included hogs could be the reason for this

inconsistency. The statistical model in the pen-mean analysis did not

include a term for the genotype x sex interaction-

DISCUSSION

Although the only significant difference in this study was that in

Table 6.11. Overall means, standard deviations and estimated differences

between Nn and NN ABRO-PTH pigs in full dissection traits, and

the relationship between carcass and rwnpback tissue composition.

Trait Overall mean SD

Difference

Nn - MN SE

Regression on rumpback tissue (%)

SE r (1

no. pigs 18 vs 18

Carcass weight (kg) 66.5 4.18

Carcass lean (%) 44.2 2.74 1.6 1.13 0.71 0.07 0.89

Carcass fat () 32.1 3.42 -1.6 1.38 0.79 0.06 0.94

Carcass bone (%) 7.4 0.63 0.2 0.20 0.29 0.07 0.70

If Residual correlation, after fitting second model in (ii).

meat colour there are reasons to suspect that the changes in lean content

reflected a true genotypic difference which could not be demonstrated with

statistical significance because of insufficient data. As shown in Table

6.15, previous investigations found an average increase of 1.5 % leanness

in heterozygotes (Nn) relative to homozygous normal (NN) pigs. The

current estimates are thus in good agreement with those findings. The

present results should also be contrasted with a difference of about 3.5 %

between the two homozygous genotypes, and with a 3.2 ¼ difference between

reactor and non-reactor pigs, as averaged from the literature (see Table-

6.15). Thus, the experimental evidence gathered so far seems to support

the opinion that, while halothane susceptibility is a recessive trait the

accompanying effects on lean content are more or less additive (e.g.

Jensen, 1981; Webb, 1981). The same would seem to apply to other effects

of susceptibility, such as those on meat colour. Nevertheless, better

estimates are still required to confirm this indications.

The question may be asked as to how some effects of the halothane

gene are recessive while others appear to be additive. The following

model, though simplistic, might provide one possible explanation.

According to Gronert (1980), the first event in the malignant hyperthermia

reaction -which characterises halothane susceptibility- is an abrupt

increase in intracellular ionized calcium triggered by two types of

stimuli (a) some anaesthetic drugs (b) muscular contractile t1v1ty The

Ca-f-f- rise causes a sharp increase in circulating catecholamines and an

elevation in general metabolism -an homeostatic attempt to reverse the

calcium elevation. Gronert (1980) suggested that the halothane locus

might code for a cell membrane enzyme controlling Ca++ movements.

Therefore, suppose that there are normal and mutant alleles, the latter

Table 6.12. Overall means, standard deviations and

estimated differences between Nn and NN

PTH pigs in amount of lean in the joints

at constant total lean weight.

Difference # Overall

Joint mean (g) SD Nn-NN SE

Hand 4859 588 186 171.8

Collar 4107 527 50 135.5

Ribback 3608 484 -225 140.5 *

Streak 3951 551 -363 219.7 *

Ham 8095 716 210 173.8

Rumpback 1881 210 132 69.5 **

# Difference between mean weight of lean in the

joint, adjusted to 26500 g total lean in the

carcass.

*P < 0.10

**p < 0.05

Table 6.13. Overall means, standard deviations and

estimated differences between Nn and NN

PTH pigs in amount of fat in the joints

at constant total fat weight.

Joint Overall mean (g) SD

Difference #

Nn-NN SE

Hand 2487 409 -102 85.6

Collar 3028 377 36 96.5

Ribback 3964 638 84 99.4

Streak 4152 644 -67 132.7

Ham 3233 432 -87 102.8

Rumpback 1463 255 77 44.8 *

if Difference between mean weight of fat in the

joint, adjusted to 21000 g total fat in the

carcass.

*p < 0.10

Table 6.14. Overall means, standard deviations, estimated regression

1

coefficients (/3) of bone in joint on total carcass bone

and estimated differences between Nn and NN PTH pigs in

amount of bone in the joints at constant total bone weight.

Joint Overall mean (g) SD Nn

Pooled SE NN of",

Difference #

Nn-NN SE

Hand 1097 148 0.30 0.22* 0.031 -49 27.5

Collar 741 101 0.08 0.16 0.049 26 42.9

Ribback 810 120 0.11 0.08 0.058 -12 50.5

Streak 386 80 0.18 0.15 0.028 -33 24.1

Ham 1087 128 0.30 0.19* 0.033 -44 29.0

Rumpback 368 59 0.01 0.08* 0.023 49 19.8

*p < 0.05

# Differences between mean bone weights in joint, adjusted to 4500 g

total bone weight in the carcass.

producing an hypofunctional -or functionless- enzyme variant. The

enzymatic activity in heterozygotes would then be intermediate. Following

an appropriate stimulus there would be a rise in intracellular Ca++, both

in mutant homozygotes and in heterozygotes; the increase, however, would

be twice as high in the former than in the latter. Thus, Ca++ levels in

heterozygotes would not reach a threshold where control of the metabolic

processes is lost and malignant hyperthermia ensues. Those levels would

only occur in homozygous mutant pigs after type (a) stimuli, or after very

intense muscular activity. Liability to malignant hyperthermia would thus

be a recessive.trait. Type (b) stimuli, however, occur often in everyday

life, during exercise or excitement. In most cases their intensity would

not be high enough to make Ca-I-I- reach a critical level; nevertheless,

they would still elicit an increase in circulating catecholamines and in

general metabolism proportional to the calcium rise. The lipolytic and

fat mobilizing effects of noradrenaline are well known (see Gregory,

1981). By enhancing glycolysis the catecholamines may also be a factor of

meat quality (Opsahi et al., 1981). More or less additive genotypic

values in traits like body leanness and meat colour would occur in this

way. Of course, similar models could be constructed with different

assumptions. The details are less important than the general features:

a primary metabolic reaction, whose kinetics is affected by varying

cnvrozertal stimuli and by a locus with gene dosage effects,

- a series of secondary metabolic reactions, whose intensity depends

on the concentration of product from the primary reaction,

- a threshold on the scale of concentration of product, after which

Page .134

Table 6.15. Summary of effects of the halothane locus on

% lean content in pigs.

Estimated Comparison difference Trait Source *

11 HP vs RN 3.2 3.8

nn vs NN 2.7 4.3 3.5

Mn vs NN

carcass lean 1 rump lean 2

carcass lean 3 carcass lean 4

mean

1.0 carcass lean 3

2.1 carcass lean 4

1.6 carcass lean this study

1.5 rump lean this study

# HP = halothane positive reactors; RN = halothane negative ractors

* References

Pool of nine estimates from the literature; see Chapter 2 (Table 2.3).

ABRO-PTH; Webb and Jordan (1978).

German Landrace; Schneider, Schworer and Blum (1980).

Danish Landrace; Jensen (1981).

the secondary reactions develop into malignant hyperthertnia.

A model with such features would determine that the property of

dominance is a function of the trait being looked at, rather than of the

locus.

The next question is whether the secondary effects are indeed

plelotropic manifestations of the halothane locus or, rather, the result

of non-random association with other genes. As discussed earlier, there

had been less than three generations of random mating following the

original Pietrain x Hampshire crosses, before PTH was divided into the SS

and SR lines. Under such conditions the - susceptibility' allele might

have 'hitch-hiked some Pietrain genes into SS. Is it possible,

therefore, that some loci, linked to the halothane locus, might affect

traits like lean content? The locus of the structural gene for the enzyme

6-PGD, which recent studies situated at about 9 centimorgans from the

halothane locus (Jrgensen, 1981), could meet the requirements. This

enzyme acts in the pentose cycle, whose function is to produce NADPH for

lipid biosynthesis. Steele et al. (1972) and Rogdakis (1974, 1979) have

demonstrated a clear relationship betweenenzyme activity in the pentose

shunt and fatness in pigs. The genetic correlation between NADPH

generating enzymes activity and backfat thickness was estimated at 0.73 ±

0.16 (Rogdakis, 1979). The gene for 6-PGD is polymorphic in the pig; two

codominant alleles produce three electrophoretic genotypes (Cahne, 1979).

If 6-PGD genotypes affect lipid synthesis in the pig as they do in

Drosophila (Cavener and Clegg, 1981) their effects will be confounded, in

populations out of linkage equilibrium, with the halothane genotypic

b

effects. Unfortunately, 6-PGD was not investigated in PTH. However, its

map distance to the halothane locus might be well below the expected

length of the linkage block remaining intact after three generations of

random mating (Hanson, 1959). Here again, as in the case of the

reproductive traits, the effects of the halothane locus might be

confounded, in the PTH lines, with those of other loci in a linkage block

around it. As was remarked for the H locus, it would be interesting to

assess the present allelic frequencies at 6-PGD in the SS and SR lines.

And yet, the present differences in leanness agree with all previous

investigations, in direction and in order of magnitude. Apart from PTH,

those investigations involved samples from the Dutch Landrace (Elkelenboom

et al., 1980), German Landrace (Schneider et al., 1980) and Danish

Landrace (Jensen, 1981) breeds. The studies did not provide information

on 6-PGD genotypes; however, it would be surprising if the halothane

gene was associated with the same 6-PGD allele in all the experiments.

In fact, population studies revealed that the halothane locus was in

linkage equilibrium with 6-PGD in the Danish Landrace breed (JØrgensen and

Hyldgaard-Jensen, 1981). On this basis it could be concluded that the

halothane locus exerts its own, additive effects on leanness. The

possibility still remains that linkage, disequilibrium with 6-PGD causes

some confusion as to the correct magnitude of the halothane genotypic

values but this hypothesis is still to be tested.

Judging the present results from a practical viewpoint, the paler

musculature in heterozygotes might, in some instances, be regarded as a

disadvantage. Paleness is one of the characteristics defining PSE -pale,

soft, exudative- pork; another is lower muscle pH (Briskey, 1964);

thus, colour and pH are included in the so-called meat quality index

used in Denmark (Barton-Gade, 1981; Jensen, 1981). Meat colour, however,

does not seem to be an economic problem in itself; a recent British study

concluded that there is no adverse consumer reaction against paler pork;

paler lean did not represent a sales problem for fresh or cured meats

(Smith and Lesser, 1982). Paleness is associated with increased drip

losses in fresh pork and with reduced brine uptake during curing, in PSE

carcasses. These might constitute disadvantages for pigmeat traders or

curers; their economic significance, however, varies widely (Month et

al., 1981; Smith and Lesser, 1982). At any rate, this study failed to

detect differences in muscle pH and the genotypic difference in meat

colour was far from reaching the magnitude of those usually observed

between PSE and non-PSE carcasses (see Smith and Lesser, 1981).

Therefore, it seems difficult that differences in meat colour such as those

found in this study might represent an Important economic disadvantage for

the heterozygotes, relative to the homozygous normal pigs.

The practical significance of the differences in tissue weight

distribution is unknown. Perhaps the genotypic differences in relative

growth of some bones are interesting to those who use prediction equations

of carcass bone based on bone content In some particular joint. One

requirement of such predictors is that their relationship with the

predicted vriab1a remains stable across different genotypes. In economic

terms, changes in tissue weight distribution are usually assumed to be

unimportant (Fowler, Bichard and Pease, 1976). In the present case it is

difficult to atribute any obvious economic significance to the differences

shown in Tables 6.12 and 6.13.

At present, the main concern of most pig breeding programmes is to

produce leaner animals. Therefore, the most important result in this

study is the indication of a 1.5 % increase in lean content in

heterozygotes. To put this effect in perspective, the annual progress in

% (rump) lean obtained by the Pig Improvement Scheme of Great Britain was

recently estimated at 0.46 and 0.87, in Large White and Landrace

respectively (Jones, 1982). In national terms, thus, the advantage in

heterozygotes would represent some 1.5 - 3.5 years of selection.

Since PTH is a synthetic experimental population the extent to which

the present results can be extrapolated to commercial pig populations may

be arguable. A further experiment has therefore been started at ABRO to

estimate genotypic values in British Landrace. In the meantime, if a

decision has to be taken on the likely benefits of developing specialised

sire lines the present results might be conveniently pooled with other

results in the literature and an economic assessment might be attempted on

this basis. Such evaluation must consider the costs incurred in

maintaining and selecting additional lines with reduced prolificacy and

with stress susceptibility problems (Smith, 1981). The set of economic

values to be assigned to the traits will vary with the circumstances; for

example: a susceptible sire line may be bred by some integrated scheme

producing and selling fresh pork; alternatively, it may be developed by a

breed society on a national scale; the economic weighting of the traits

is likely to vary between these two alternatives. Because of simpler

organisational problems it would seem more feasible, in principle, to

exploit the halothane gene by means of specialised lines in an integrated

production scheme, than in a general breeding program of national

proportions (Smith, 1981). However, as pointed out by Webb (1981), the

Theterozygote mating system is already in use in some European countries,

with sires from halothane susceptible type of breeds, such as Pietrain or

Belgian Landrace, mated to halothane tolerant type of darn breeds, such as

Large White x Landrace.

CHAPTER 7. CONCLUDING REMARKS

The studies in the preceding chapters have dealt with diverse aspects

of the halothane susceptibility phenomenon in pigs. In two instances they

utilized results from experiments performed with specific aims at ABRO:

to assess the effects of age on susceptibility incidence and to estimate

genotypic values for growth and carcass traits. The rest of the studies

relied on field data resulting from the development of halothane

positive and negative experimental stocks. In general, most of the

results can not be regarded as conclusive. Instead, they provided

indications which, together with some speculation, might hopefully serve

as a framework for testing hypotheses in future investigations. In this

chapter these will be briefly reviewed with the aim of outlining some

problems for future research.

(i) The mode of inheritance of susceptibility.

In Chapter 3 the single—recessive hypothesis was rejected as the mode

of inheritance in British Landrace. While this is the first time this

hypothesis was relected it also seems to have been one of the few

occasions when it was formally tested against some rival hypothesis. The

study was prompted by the observation that penetrance was unusually low in

Landrace; furthermore, it seemed to vary between families.

However, the Landrace data were not entirely adequate for testing

genetic hypothesies because: (a) the population was a mixture, its

probability structure required nuisance parameters which rendered the

conclusions of the analysis conditional on their value; and (b) there

were no matings between reactors and non-reactors. Ideally, inferences

about the mode of inheritance of any trait should be based on the outcome

of all possible mating types. Hence, these results should be seen as

preliminary indications that a single and strictly recessive mode of

inheritance may not be appropriate in British Landrace. These pigs are

thus interesting for future investigations, as they offer the possibility

of discovering new aspects of halothane susceptibility. There are also

practical implications since the breed is widely exploited in Great

Britain. Clearly, the mode of Inheritance has to be satisfactorily

elucidated before the benefits of alternative breeding policies can be

fully assessed.

It thus seems Important to verify these findings, which can be

summarised as follows: under a single-locus model with two alleles 91 %

of one of the homozygotes and 22 % of the heterozygotes appeared to be

reactors in British Landrace. This was in contrast with the findings in

Pietrain/Hampshire, where susceptibility appeared to be strictly

recessive.

Irrespective of what genetIc hypotheses are to be tetcd in future

research, it is now clear that with halothane susceptibility the classical

Mendelian segregation ratios tend to be distorted by differential

mortality and incomplete penetrance. The problem, therefore, is one of

estimation of parameter values in genetic models. The adequacy of

different hypotheses about the parameter values has to be judged in

probabilistic terms. In these circumstances the general method of genetic

inference called segregation analysis (Morton, 1969), which is based on

the concept of likelihood, would seem to be the most appropriate

analytical tool. Hypotheses can be tested by standard likelihood ratios

(Edwards, 1972).

What general models could be used? A single-locus with two alleles

could provide the first framework. Hypotheses should concern the value of

penetrance in heterozygotes, that is, whether it is different from zero

(recessivity) or from unity (dominance). If an intermediate penetrance

for the heterozygotes is confirmed curiosity will then demand the testing

of new hypotheses. After all, how could it be that this trait sometimes

behaves dominantly, sometimes recessively ? New hypotheses will in turn

require some extension of the single-locus-two-alleles model. Multiple

allelism is a possibility under a single-locus model. Otherwise the

number of loci can be increased. Simon (1980) suggested that the

multifactorial-threshold model could be an alternative. However, a good

amount of evidence shows that there is a locus with major effects on

halothane susceptibility in a well studied linkage group in pigs (e.g.

Andresen, 1981; Jrgensen, 1981). Thus, models keeping a -major locus

but involving some genetic modifier device seem a more realistic option.

The two-locus-suppressor niüdi of Chapter 3 Is one such model. It

allowed rejection of the single-recessive and -dominant hypotheses in

Landrace and also showed it could improve the single-recessive hypothesis

in Pietrain/Hampshire. However, some alternatives can be envisaged, which

werenot compared with the two-locus-suppressor model. For example,

genotypes at the second locus might confer, or fail to confer,

protection against halothane reaction on the heterozygotes at the

susceptibility locus. The modifier effects could arise from polygenic

variation rather than from gene subtitutions at a single locus, as in the

mixed models of Morton and MacLean (1974). Some of these variants could

be better than the two-locus-suppressor model. New developments should be

taken into account when modelling; thus, the finding of Chapter 5 that

incidence was higher in females than in males could be featured in various

ways; for example: the suppressor gene could be X-linked rather than

autosomal in the two-locus model of Chapter 3. Progress in the field of

biochemistry of halothane susceptibility should be followed closely; the

more that is known about the underlying biochemistry the more realistic

these models are likely to be. In any case, whatever general model is

adopted it should, at this stage, be constructed in a way such that by

specific restrictions at some of Its parameters it returns the primary

single-recessive (or -dominant) hypothesis.

What kind of data are required ? The answer depends on the genetic

hypothesis to be tested. For example: it could be possible to test the

hypothesis that penetrance is partly controlled by an autosomal recessive

suppressor by Intermating non-reactor offspring from reactor x reactor

matings. Among the progeny there should be entire litters of non-reactors

(double homozygotes nnss; see Table 3.1); when intermated these pigs

should always breed non-reactors. When mated to reactors they should

yield only reactors or reactors and non-reactors in a 1:1 ratio, depending

on the genotype of the reactor parent at the suppressor locus. However,

at this stage It seems more interesting, and indeed logically neccessary,

to try a variety of genetic models using segregation analyses, as

discussed above. For this purpose a set of halothane testing results from

reactor and non—reactor parents and their offspring constitutes the basic

experiment advocated here.

The prior probability structure of this population should be as simple

as possible, not only for computational economy but also because it is

desirable that statements about the value of parameters of interest in the

model can be made without having to invoke the value of nuisance

parameters. Thus, if a population is set up after mixing pigs from

different subpopulations or lines, the parental group should come from the

F2 or later if models are not to include such nuisance parameters as

variances and covariances of gene frequency differences among lines.

Mating should be random in the grandparental generation and all possible

mating combinations between reactors and non—reactors should be

accomplished in the parental group. Ideally, all pigs should be kept on

the same farm.

At this point it is convenient to recall some results from previous

chapters. In Chapter 4 it was discussed how, given certain assumptions,

the mode of inheritance of susceptibility can be an artifact of the

duration of the halothane test. It was also found that about 20 %

positive reactions in a 5—minute test occurred after the .third minute in

British Landrace. Thus, there is evidence showing that the standard

3—minute test is too short to detect the slower reactors of this breed.

It might be rewarding to extend the test duration as much as possible

Reaction time can also enter into models for the inheritance of

susceptibility in several ways, as discussed in Chapter 4. It was shown

in Chapter 5 that the incidence of susceptibility changes markedly with

the age of the pigs. If the complication of treating penetrances as

Page l'5

functions of age is to be avoided, all pigs in the experiment, parents and

offspring, should be tested at an uniform age which, according to findings

in Chapter 5, should not be earlier than five weeks.

A multi—purpose data set derived from such an experiment would be

very useful at this stage, when there is a need not only of verifying the

specific findings of Chapter 3, but also of exploring a variety of genetic

models. Once a model has been chosen it can be validated further by

testing some of its consequences in ad hoc experiments.

(ii) Changes in productivity traits associated with susceptibility.

There is now a large amount of evidence showing what traits of

economic importance are likely to be affected by halothane susceptibility.

Moreover, the direction of these changes and, in some instances (e.g.

lean content), the order of magnitude are also reasonably well

established. Nevertheless, there are still a number of problems that

require elucidation.

The question of whether these effects are pleiotropic manifestations

of the halothane locus or the consequences of linkage disequilibria with

other closely linked loci has been discussed in Chapters 2 and 6. It

should suffice here to stress the point that results in the literature

call for a thorough assessment of the effects of genotypes at the H and

6—PGD loci on reproduction and leanness respectively, independently of,

and also interacting with, genotypes at the halothane locus. A number of

very interesting genetic problems could arise from a situation where two

loci in a relatively short chromosomal tract are found to have effects on

the same metric trait.

Another question springs from the findings of Chapter 3 on the mode of

inheritance of susceptibility: if there is genetic variation modifying

gene action at the susceptibility locus, how will it affect the secondary

effects of this locus ? The question was proposed by Webb (1981) in a

slightly different form : could it be possible to separate harmful

effects, associated with stress susceptibility, from beneficial effects,

by exploiting modifier genetic variation ? In principle, the possibility

that genetic modifiers could act partially, modifying only some aspects

of susceptibility leaving others unchanged, seems unlikely. Harmful and

beneficial effects, like stress susceptibility and increased leanness, are

seen here as closely related manifestations of a single biochemical or

physiological phenomenon. However, this is a matter of empirical

investigation, which can be tackled once the problems raised in Chapter 3,

concerning the mode of inheritance ; are clarified If there are lcd

whose alleles are modifiers of gene action at the susceptibility locus

selection for and against halothane reaction will change their allelic

frequencies. In this case, how adequate are selected experimental lines

as models of commercial pig populations ?

It would probably be generally agreed that much of what is known so

far about the effects of halothane susceptibility on other traits can be

branded rough knowledge'. For example, evidence is beginning to gather

showing that susceptible females produce smaller litters at weaning. This

seems to be due to reduced prolificacy and higher piglet mortality during

lactation. But there are no precise details. How could the differences

in prolificacy arise ? Do they result from reduced ovulation rate, higher

embryo mortality or both ? And what are the causes of deaths among

piglets ? Is agalactia more frequent in susceptible females ? Do they

trample piglets down more frequently ? Are there differences in gestation

length ? Are the piglets weaker ? and so on. It is evident that there

is no shortage of questions concerning the effects on reproductive traits.

Fortunately, some of them can be answered without much difficulty, by

simple and careful collection of the relevant data, after randomization of

some environmental influences.

A similar argument applies to the effects on carcass traits.

Susceptible pigs appear to have heavier, leaner and shorter carcasses and

paler meat. These are the statistical facts. But how can they be

interpreted in biological terms ? It Is suspected that catecholamlnes may

be involved In the determination of differences in leanness and meat

colour (Gregory, 1981) but this is still to be clearly established. What

other endocrine systems are Involved ? In general terms, there is still

much to learn about the physiological bases of the changes in reproductive

and carcass traits that accompany halothane susceptibility. Such

knowledge could throw light on the possibility of modifying some of the

undesirable effects of susceptibility by means of background genetic

variation.

In general terms, most problems concerning the genetics of halothane

susceptibility in pigs have so far been approached on a statistical level.

Although there is still a need for more and better estimates, particularly

in relation to reproductive traits, this approach has already produced

useful information which nowadays enables breeders to make some judgement

of the economic balance between expected beneficial and harmful. effects.

However, a growing list of unanswered questions now requires an attack on

physiological and metabolic grounds. This will give geneticists a better

understanding of the halothane susceptibility trait, and could also yield

information of potential usefulness for practical breeding. Eventually,

such investigations may turn this genetic system into a candidate for

manipulation by means of genetic engineering techniques.

APPENDIX

Halothane testing results in Pietrain/Hanipshire:

Positive x Positive matings Negative x Negative matings

Sire Darn No.Progeny No.Positive

1 1 8 8 2 6 6

2 3 7 6 4 7 7 5 8 5

3 6 3 3 7 1 1 8 4 4

4 9 6 6 10 8 8 11 3 3

Positive x Negative matings

1 12 8. 8 4 13 10 0

14 7 3 15 8 0 16 7 0

Negative x Positive matings

5 17 11 9 18 6 6

6 19 8 8

Sire Dam No.Progeny No.Positive

6 20 7 4 21 9 0

7 22 8 1 23 6 .2 24 9 0

8 25 7 1 26 6 0 27 2 0

9 28 6 0 29 6 0 30 5 1

10 31 2 0 32 7 2

11 33 6 0 34 4 0 35 6 2

12 36 9 0 37 7 0

13 38 6 1 39 9 2

1.4 40 9 3

Halothane testing results in Landrace:

Positive z Positive matings

Sire Dam No.Progeny No.Positive

1 1 12 7 2 3 3

2 3 9 9 3 4 10 . 7

5 10 3 6 7 2 7 2 1

4 8 11 10 5 9 7 2 6 10 5 0

11 5 5 7 12 6 4

13 4 3 14 7 6 15 1 1

8 16 6 3 17 7 4

9 18 9 4 19 10 9 20 11 9

10 21 9 4 22 5 4 23 9 6

11 24 6 1 12 25 4 4 13 26 7 6

27 6 6 14 28 10 10

Negative x Negative matings

Sire Darn No.Progeny No.Positive

15 29 9 2 30 4 0

16 31 6 5 32 8 0

17 33 10 0 34 3 0

18 35 7 0 36 7 1

19 37 1 0 38 12 0 39 3 0

20 40 8 0 41 5 0 42 4 0

21 43 6 . 2 22 44 7 0

45 11 2 46 6 0

23 47 6 0. 48 5 0 49 4 0

24 50 6 0 51 9 2 52 7 0

25 53 6 0 26 54 10 0

55 9 0 56 5 0

27 57 5 0 28 58 6 0

59 10 0 60 9 0

ACKNOWLEDGEMENTS

This thesis was done under the joint supervision of Dr A. J. Webb

and Dr W. C. Hill. Above all I wish to express my deep gratitude to

them for their generous support, constant encouragement and invaluable

guidance during the course of my studies. Dr Webb made available the

data, for which I am particularly indebted.

I should like to express my appreciation to Professor J. W. B. King

for providing excellent facilities at the ARC Animal Breeding Research

Organisation. Dr C. Smith read Chapters 3 and 6 and provided careful and

constructive criticism which saved me from some errors. Professor A.

Robertson gave me valuable suggestions on the problem of random genetic

differentiation of lines. I am most grateful to them.

Eduardo Avalos read and criticised the manuscript. I owe a great debt

to him for his constant friendship and support. I am also indebted to

Andy Sutherland who clarified to =c many statistical problems and placed a

battery of useful programs at my disposal.

I would also like to thank:

Mr D. I. Sales and Mrs C. McCoubrey for statistical advice; Mr D.

Nicholson, Mr D. Maxwell and Mr R. Findlay for their guidance in the use

of computing facilities and for their help in debugging many programs;

Anne Douglas and Maureen Edwards for computational assistance; Mr I.

Will and staff at ABROs Mountmarle farm for collecting most of the data

for this work and Miss H. MacLean for data preparation.

I wish to express my great obligation to CONICET and INTA, from

Argentina, for their financial support. Of my colleagues at INTA

Pergamino I must mention Mr Manuel Bonino for his faithful support over.

the years.

Finally, I wish to express my special gratitude to my wife, Nora, and

our children Natalia, Pablo and Cintia, for their love, encouragement and

patience, and to my parents for their constant support.

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