Courtesy Australasian Pig Science Association Reproduced from: 2001 Manipulating Pig Production VIII The Eight Biennial Conference of the Australasian Pig Science Association was held in Adelaide, South Australia, 25th November - 28th November 2001. The Proceedings, 'Manipulating Pig Production VIII', contains 5 Reviews and 3 Symposia, presented by leading scientists from Europe, North America and Australia, and 86 one-page papers. All papers were reviewed by external referees. Reviews Productive research environments in the rural industries - can we do better? by Professor David Lindsay; Factors limiting the performance of growing pigs in commercial environments by Dr John Black et al.; Australian and international developments in the assessment and management of piggery odours by Dr Peter Watts et al.; The prevalence and eradication of pig diseases in Australia by Dr Ross Cutler; Luminal bacteria and regulation of gut function and immunity by Dr Denise Kelly and Dr T.P. King Symposia "Managing the eating quality of pork" was convened by Dr Mike Taverner and included the following: »» Managing the eating quality of pork - what the producer can do by Dr. Darryl D'Souza and Dr B.P. Mullan »» Managing the eating quality of pork - what the processor can do by Ms Heather Channon »» Managing the eating quality of pork - realizing benefits along the value chain by Dr David Meisinger 'Pig AI in Australia - Opportunities and limitations' was convened by Dr Paul Hughes and included the following: »» Production of fertile insemination doses by Dr Billy Flowers »» Estimating the optimum timing of insemination in pigs by Dr Paul Hughes and Dr G. Pope »» Effects of the inseminator and insemination type on efficacy of AI by Dr Billy Flowers »» Biosecurity in AI centres in Australia - should we be concerned? by Dr Ranald Cameron 'Understanding the nutritional chemistry of grains will help to improve the profitability and sustainability of the pig industry' was convened by Dr John Pluske and included the following: »» Comparative digestion of energy from grains fed to pigs, poultry and ruminants: Can the efficiency of pig production be improved? by Dr Rob van Barneveld et al.; »» Light microscopic techniques to understand starch digestibility by Dr Karin Autio; »» How effective are supplemental enzymes in pig diets? by Dr Mingan Choct and Mr. D.J. Cadogan One-page papers Papers were presented and published on the followings themes : Animal Health (6 papers); Genetics & Animal Breeding (8); Growth & Development/Physiology (15); Housing & Environment (7); Meat Quality & Meat Hygiene (18); Nutrition, Nutrient Intake, Nutrient Quality & Utilization (24); Reproduction (8); ORDER DETAILS Edited by P.D. Cranwell. Hard-cover, 312 pages. ISBN 0 957 7226-1-3. Published by APSA, Werribee, Victoria 3030, Australia. Price: A$100 (plus P+P) for overseas and A$110 (plus P+P) for Australian customers. VISA and Mastercard accepted. Order forms are available for Australian and Overseas customers.

Introduction

Over the last ten years the introduction of 'New-Fashioned Pork' in keeping with the increased consumer health awareness has seen the overall fat content of pork reduced by 60-65% ¹. In addition, comparisons between cooked meats have indicated that 'New-Fashioned Pork' is not only significantly lower in fat compared to the leanest cuts of beef and chicken but also represents better nutritional value compared to other major animal protein products¹. The National Heart Foundation Food Approval Program currently approves 14 of the 22 cuts of 'New-Fashioned Pork' ².

In order to achieve this the Australian pork industry has had to implement significant changes to its production systems to produce leaner and 'healthier' pork. This has seen the introduction of leaner genetic lines, improved feed formulations, the use of in-feed antimicrobial agents, metabolic modifiers to improve carcass leanness, entire male pigs instead of castrated male pigs which are fatter, and heavier slaughter weights. Yet, the increase in pork consumption in more recent years has for the most part been minimal³.

Against a backdrop of increased global pork consumption in comparison with other meats, per capita pork consumption in Australia still lags well behind countries in North America and Europe ⁴, countries that in many instances have similar demographic profiles to Australia. Overseas research has shown that changes to production systems similar to that adopted by the Australian pork industry have had a negative impact on the many quality attributes of pork and this may explain why pork consumption in Australia has not increased significantly over the last decade.

Eating quality of Australian pork

A study conducted in 1994 by the Australian Meat and Livestock Corporation comparing beef, lamb, pork and chicken found that only 15% of consumers considered pork to be tender and juicy, whilst 32% of consumers stated that pork was inclined to be dry⁵. Consumers also rated pork as being the toughest and driest of all meats assessed.

In 1997, a survey of Melbourne butchers and supermarkets identified considerable variations in pork tenderness, with 54% of pork loins purchased reported to be unacceptably tough⁶. An "Eating Quality Assurance for Pigmeat" feasibility study (1997) found that consumers consider pork to be tough and dry in comparison to other meats due to inadequate intramuscular (IM) fat, the high incidence of pale, soft, exudative (PSE) pork, cold shortening (shortening of muscles due to over-effective chilling of hot pre-rigor carcasses), inadequate ageing and overcooking⁷.

The study also identified boar taint in fresh and processed pork as the most significant cause of consumer complaints. The above studies quite clearly emphasise that consumers do not rate the eating quality of Australian pork highly in comparison with other meats.

Production factors affecting eating quality of pork

Pork eating quality is characterised by sensory or palatability traits such as tenderness, juiciness, flavour and aroma⁸ and can be influenced by a range of production and processing factors. This paper will focus on the production factors with the emphasis on what pork producers can do to improve the eating quality of Australian pork.

Genotype

Halothane gene

Pigs carrying the Halothane (Hal) gene (homozygous recessive - nn (reactors); heterozygous - Nn (carriers) are leaner, grow faster and have better carcass conformation compared to 'normal' (homozygous dominant - NN) pigs⁸.

However, the introduction of the Hal gene in pig breeds has coincided with an increase in the incidence of pork quality defects such as PSE pork⁸. Pigs carrying the Hal gene (nn and Nn) are stress susceptible (Porcine Stress Syndrome) due to disorders in muscle Ca2+ regulation, which results in the muscle being hypersensitive to stimulation by various stressors⁹. Conditions during transport and at the abattoir that cause no stress to 'normal' pigs can stress Hal pigs causing a rapid rate of pH decline post-slaughter while muscle temperature is still high (>36°C), resulting in PSE pork¹⁰.

Pale, soft, exudative pork is characterised by its unusually pale colour, soft or sloppy texture and excess exudation¹¹. Carcasses exhibiting PSE pork carcasses also tend to have a higher incidence of colour variations between portions of the same muscle (especially in the hams) and between adjacent muscles of PSE carcasses; this is referred to as 'two-toning' ¹².

Consumers are hesitant to purchase pork that is pale or 'two-toned' and relate such pork to be of inferior eating quality. Most studies also indicate that PSE pork has inferior eating quality with reduced tenderness and juiciness¹³. Studies have also shown that the improvements in tenderness associated with ageing of pork are not applicable to PSE pork¹⁴.

While the Hal gene is not wholly responsible for the high incidence of PSE pork in Australia, a survey of major pork processors in Australia found that the incidence of PSE was 51% ¹⁵. A national program implemented in Australia has seen a dramatic decrease in the incidence of soft, exudative pork to 38% ¹⁶. This, however, is still high and PSE remains a major problem affecting the eating quality of Australian pork.

More recently, pork producers are becoming increasingly aware of the link between genetic lines carrying the Hal gene and the incidence of PSE pork. As a consequence, major pig breeding companies are selecting for carcass leanness and using fast growing pig genetic lines that do not have the Hal gene. A recent survey in Western Australia indicated that majority of producers supplying pigs for export were using genotypes that did not have the Hal gene¹⁷.

Hampshire gene

The Hampshire gene or the 'Rendement Napole' (RN) gene is a dominant gene and was first found in two commercial pig bloodlines in France¹⁸. The RN gene was found at a higher frequency in Hampshire pigs compared to other breeds such as Large White, Landrace, Yorkshire and Peitran. Pigs carrying the RN gene were found to have significantly higher muscle glycogen concentrations (70%) compared to 'normal' pigs¹⁹. As a consequence RN pigs exhibit an extended period of muscle pH decline post slaughter leading to an extremely low ultimate muscle pH and water holding capacity, rather than the increased rate of pH decline and protein denaturation observed in PSE pork¹⁸. While the RN gene reduces muscle pH and water holding capacity of pork, the RN gene has been shown to improve pork tenderness and juiciness²⁰. The incidence of the RN gene in Australian pig herds is unknown. However, as the number of Hampshire herds in Australia is low, it may be assumed that the impact of the RN gene on the eating quality of Australia pork is minimal. However, a detailed study will be needed to quantify the incidence of the RN gene in Australian herds and its potential influence on the eating quality of Australia pork.

Breed

Duroc and intramuscular fat

It is generally accepted that higher levels of intramuscular fat or marbling in pork have been shown to positively influence the juiciness, tenderness and flavour of pork⁸. This relationship is by no means clear-cut, with some studies reporting no effect of marbling on the eating quality of pork²¹. The production of leaner pigs in Australia over the last 30 years has seen marbling levels at <1% ²², which is below the levels of 2 - 2.5% required for optimal eating quality of pork. This has coincided with deterioration in the eating quality of Australian pork.


Comparative consumer studies indicate that meats such as chicken, beef and lamb that are considered to have better eating quality than pork⁵, also have significantly higher levels of marbling (Table 2).

Fast growing 'white' European pig breeds (Large White, Landrace, Yorkshire) have lower levels of marbling compared with the darker skinned breeds, such as Duroc^5,23 and Berkshire²³.

The Meat and Livestock Commission, UK, evaluated the Duroc breed and found that tenderness of pork was improved in pigs with Duroc gene proportions above 50% while juiciness increased when the proportion of Duroc genes was increased to 75% ²⁴. Similar improvements in juiciness, tenderness and flavour were also reported by the National Genetic Evaluation Program²³. In addition, Candek-Potoker et al. (1996) reported that pork from Duroc pigs not only had higher marbling levels but also better colour and texture compared to Large White and Landrace pigs.





Studies also indicate that the inclusion of Duroc bloodlines in predominantly 'white' European breeds can also result in improvements in pork eating quality. Recent studies conducted in Western Australia, indicate that Large White x Landrace x Duroc crossbred pigs with a high proportion of Duroc genes (50%) had higher marbling values, better juiciness, tenderness and flavour compared to pork from Large White x Landrace x Duroc crossbred pigs with a low proportion of Duroc genes (<25%)²⁵.

Sex

Most Australian pig producers moved away from castration of entire male pigs about 30 years ago to harness the production benefits associated with entire male pigs. As a consequence 10 - 15% of entire male pigs produce pork that has a perspiration or urine like smell or 'boar taint' when cooked²⁶. The major compounds responsible for boar tainted pork are androstenone and skatole²⁷. Research conducted by the Meat and Livestock Commission (1989) found that while tenderness, juiciness and flavour was unaffected by sex, abnormal pork odours were higher in pork from entire male pigs.

The export of Australian pork to Asia, which only accepts pork from female and surgically castrated pigs, has meant that the likelihood of domestic consumers purchasing boar-tainted pork has also increased. Recent results indicate that pork from males pigs castrated by using the immunological castration vaccine Improvac® had lower androstenone and skatole concentrations, higher marbling levels and lower surface exudate compared to pork from entire male pigs²⁸. Although the effects of boar taint have not been as conclusive with consumer taste tests as with objective assessment of pork quality, D'Souza et al. (1999b) reported that pork from entire male pigs tended to have poorer odour compared to pork from surgical and immunological castrates (Table 4).

Pork from surgical castrates has also been reported to have superior eating quality compared to pork from female pigs. Unruh et al. (1996) reported that at 127 kg live weight, pork from the Longissimus muscle from surgical castrates had more visible marbling, less moisture exudate and less thaw loss compared to pork from female pigs.



While many Australian consumers may not find boar taint objectionable, it appears from anecdotal observations that most of the Asian populations in Australia and overseas discriminate against pork from entire male pigs. This concern has prompted a number of producers in Australia to move towards castration of entire male pigs using Improvac®. Currently in Western Australia, Improvac® pigs are marketed under the brand name Flavasure^TM that guarantees pork to be free of boar taint.

Feeding regimes

Ad libitum feeding

The benefits of ad libitum versus restricted feeding in terms of growth performance and carcass quality are significant. Pigs fed ad libitum during the grower and finisher phase (30 - 100 kg live weight) grow faster²⁹. The ad libitum feeding of protein deficient diets 30 days prior to slaughter improved the eating quality of pork by increasing marbling levels in the loin³⁰. The increase in marbling reported by Cisneros et al. (1996) however, was also accompanied by an increase in carcass fatness. Studies have also shown that feeding high energy diets, especially in the latter stages of growth, can elevate the rate of protein synthesis and degradation which may accelerate post mortem proteolysis and improve the tenderness of pork³¹.

Dietary fats

The trend towards leaner pigs has also resulted in problems with the quality of fat in the carcass that can affect both the appearance and eating quality of pork. Problems with fat quality include soft or 'floppy' fat, fat and lean tissue separation, and flavour taints³². The amount of fat in pig carcasses increases rapidly during growth, originating both from the diet and from synthesis. Feeding diets high in unsaturated fats was found to increase the concentration of unsaturated fatty acids in carcass fat resulting in soft fat³³.

The problem of fat and lean tissue separation is often called 'lacy' fat because of its appearance in pork products such as bacon rashers. Lacy fat results from the strands of connective tissue that separate both between the fat layers, and between the fat and lean tissues during further processing of pork cuts³⁴. The cause of fat separation, unlike soft fat, appears to be due to the immature nature of the adipose connective tissue rather than to the degree of unsaturated fatty acids present in adipose tissue³⁴.

The inclusion of fishmeal in pig diets above 5%, has an adverse effect on the eating and keeping qualities of pork and pork products³⁵. While fresh pork from pigs fed fishmeal may be free of 'fishy' flavours and odours, almost invariably, stored processed products such as bacon and ham, will show unacceptable levels of rancidity particularly after freezing. Researchers from a number of countries have recommended that diets should contain no more than 5% fish meal or 0.5% fish oil and that they should not be fed within two weeks of slaughter³⁶. Because of cost, the use of fishmeal or fish oil in pig diets is not a common practice in Australia. However, these oils may be included in feeds inadvertently through other sources such as inclusion of waste cooking oils.

Feed additives

Magnesium

Magnesium (Mg) has a relaxant effect on skeletal muscle and has been shown to depress skeletal muscle activity by antagonising calcium, which is required for neurotransmitter release and muscle contraction. This reduces the secretion of neurotransmitters by motor-nerve impulses, which in turn reduces neuromuscular stimulation³⁷. Studies have since shown that dietary magnesium supplementation alleviates the effects of stress by reducing plasma cortisol, norepinephrine, epinephrine and dopamine concentrations³⁸. Consequently, studies have been conducted to investigate the influence of dietary Mg supplementation on reducing the effects of stress and improving pork quality.

Numerous studies have shown that dietary Mg supplementation in pigs resulted in improved pork quality. Otten et al. (1992) examined the use of long-term dietary Mg supplementation and reported slight improvements in pork colour and initial pH, whereas Schaefer et al. (1993) studied short term dietary magnesium supplementation and reported reduced initial pH and % drip loss. D'Souza et al. (1998) have shown that dietary magnesium aspartate supplementation at 3.2 g elemental Mg for 5 days pre-slaughter significantly improved pork quality in pigs by reducing the drip loss and improving pork colour and muscle pH (Table 5).

The effects were such that there were no PSE carcasses in the magnesium supplementation treatment groups, irrespective of the type of pre-slaughter handling³⁹. Dietary supplementation using inorganic Mg sources such as MgSO4 and MgCl2 ⁴⁰ and magnesium mica⁴¹ have also been shown to reduce drip loss, improve colour and reduce the incidence of PSE pork. The use of dietary organic magnesium supplementation as a viable method to improve meat quality in pigs was validated under commercial conditions in Victoria, Australia (Hofmeyr et al., 1999). In this study, organic magnesium supplementation significantly reduced the incidence of soft, exudative pork in all three replicates.



Although the use of dietary magnesium supplementation has been shown to improve objective measures of pork quality, there are no data relating the improvements in objective measures of pork quality with improved pork eating quality. However, as the relationship between low water holding capacity and increased meat toughness is well established⁴², it is reasonable to assume that the reduced drip loss and the lower incidence of PSE observed with dietary Mg supplementation could have a positive influence on the eating quality of pork.

Vitamin E

Dietary vitamin E (all-rac-a-tocopheryl acetate) supplementation has been shown to reduce drip loss, improve colour stability and reduce the off-flavours of both fresh and processed pork products⁴³. While dietary vitamin E supplementation can have a positive impact on the eating quality of fresh pork, the real benefits are the improved lipid and colour stability and improved water holding capacity in pork products which require frozen storage for extended periods. There are a range of factors that contribute to the deterioration in pork quality and loss of shelf life as a consequence of lipid oxidation occurring in pork and pork products. These factors include the state and content of pro-oxidants such as iron and myoglobin; the level of antioxidants such as a-tocopherol and enzymes such as glutathione peroxidase, superoxide dismutase and catalase present in muscle; the composition and amount of muscle lipids; and the storage conditions of meat and meat products⁴².

Asghar et al. (1991) and Monahan et al. (1992) attributed the reduced drip loss and improved colour stability of pork from pigs fed vitamin E supplemented diets to a reduction in lipid oxidation in muscle cell membranes. The reduced lipid oxidation in muscle cell membranes reduced the movement of water across the cell membrane post-slaughter. Duthie et al. (1989) however, attributed the reduction in drip loss and colour stability to alterations in muscle cell membrane permeability to calcium resulting in a reduced rate of glycolysis and muscle pH decline. If so, then the increased oxidative stress encountered by Hal pigs (nn and Nn) may be alleviated by dietary vitamin E supplementation. However, Warner et al. (1995) found that feeding vitamin E to Hal pigs did not prevent the PSE condition and had no major effect on the water holding capacity of pork.

Metabolic Modifiers

Porcine somatotrophin

Intramuscular administration of porcine somatotropin (pST) is an effective management strategy to reduce backfat in pigs⁴⁴ and is one that is quite widely used in Australia. Porcine somatotrophin increases protein deposition and decreases subcutaneous, intermuscular and intramuscular fat deposition resulting in leaner carcasses⁴⁵. Studies by Lefaucheur et al. (1992) and Ender et al. (1992) have reported reductions in both carcass fat and marbling levels in pork from pigs administered pST without any detrimental effects on the eating quality of pork. The above studies (Lefaucheur et al., 1992; Ender et al., 1992) are in contrast to that reported by D'Souza et al. (2001) who found that pork from pigs administered pST had lower consumer preference scores for tenderness, juiciness and overall acceptability. Solomon et al. (1990) reported that pST administration increased muscle fibre size and subsequent shear force, an objective measure of tenderness, of fresh pork. The use of pST has also been reported to reduce calcium-activated proteolysis in the Longissimus muscle, thereby preventing improvements in tenderness during the ageing process⁴⁶.

Porcine somatotrophin administration remains an important management strategy enabling Australian pork producers to better meet the demands of the consumers for leaner pork. However, the Australian pork industry must pay additional attention to the potential negative influence of such management strategies on the eating quality attributes of pork.

Chromium

Chromium supplementation in pig diets was found to improve feed efficiency, lean meat yield and reduce backfat thickness⁴⁷. However, limited research has been conducted on the effects of dietary chromium supplementation on pork quality. In a recent study by Matthews et al. (1999), dietary chromium proprionate supplementation in finisher pigs increased the level of marbling and reduced the purge loss in the loin muscle. However, the reasons for the increase in marbling and the reduced purge loss are unknown. In contrast, O'Quinn et al. (1998) reported that the use of chromium picolinate supplementation had a detrimental effect on pork colour. As it is possible that the source of chromium may be responsible for the varied effects of chromium supplementation on pork quality, these effects should be further investigated. Dietary chromium supplementation is another management strategy to reduce carcass fatness that is widely used by Australian producers. While the research to date has yet to elucidate the effect of chromium supplementation on the eating quality of pork, early indications are that it may have a negative impact.

Betaine

Betaine is an active methyl donor with a lipotropic effect⁴⁸. Adding betaine to pig diets has shown to reduce and change the distribution of carcass fat⁴⁹. Xu et al. (1999) reported that betaine supplemention resulted in improved carcass quality and improved subjective colour and marbling scores. In contrast, Øverland et al. (1999) reported no effect of betaine supplementation on objective meat quality. While the effects of dietary betaine supplementation on objective pork quality are varied, there is no information regarding the effects of dietary betaine supplementation on the eating quality of pork.

Conjugated linoleic acid

The use of dietary conjugated linoleic acid (CLA) supplementation has also been shown to be an effective management strategy in reducing backfat in pigs⁵⁰. Studies by Dunshea and Ostrowska (1999) and O'Quinn et al. (1998) found that dietary CLA supplementation had no effect on objective pork quality measures such as surface lightness, muscle ultimate pH, drip loss or cooking loss. Dietary CLA supplementation in pigs was also reported to increase marbling levels⁵¹. However, D'Souza et al. (2001) reported that pork from control pigs had better eating quality compared to pork from CLA fed pigs. The influence of dietary CLA supplementation as an effective strategy to reduce carcass fatness is well established. However, the impact of dietary CLA supplementation on the eating quality of pork is somewhat less clear-cut and this warrants further investigation.

Ractopamine

Ractopamine is a b-agonist that increases the protein deposition rate in pigs⁵². Ractopamine has recently been registered as a feed additive to improve carcass leanness (Paylean®) for use in the United States of America and is currently under consideration for registration in Australia. Dietary ractopamine supplementation had no effect on muscle pH, colour, water holding capacity or marbling levels. Smith et al. (1995) reported that loin muscle from female pigs had higher muscle pH, lower cooking loss and darker colour, while that from entire male pigs fed ractopamine had higher drip loss and paler colour. A negative effect of ractopamine on objective measures of tenderness was also reported by Uttaro et al. (1993).

Conclusion

There is tremendous opportunity for the Australian pork industry to build on its export market in Asia and at the same time increase its domestic market. However, significant steps must be taken to consistently produce pork that is lean, tender, juicy and free of unpleasant flavours and aromas. The production factors described in this paper indicate that producers can go a long way to improving the eating quality of Australian pork. The use of pigs with a high proportion of Duroc bloodlines, castration of entire male pigs and the elimination of the Hal gene from Australian pig herds are just some of the ways pig producers can influence the eating quality of pork. However, the implementation of such changes will add to the cost of production especially if backfat thickness and carcass weight continue to form the basis of payment to pig producers in Australia. Therefore, the Australian pork industry must look to larger pork producing countries such as USA, Denmark and The Netherlands and adopt their 'whole industry approach' to pork production and implement similar eating quality standards as part of an overall quality assurance system to produce pork that is lean, tender, juicy and free of unpleasant flavours and aromas.

References

1. Barnes et al., 1996
2. Heart Foundation, 2001
3. ABS, 2000
4. AMI, 1999
5. AMLC, 1994
6. Hofmeyr, 1998
7. Bennett, 1997
8. Wood, 1993
9. Fuji et al., 1991
10. Briskey and Wismer-Pedersen, 1961
11. Bendall and Wismer-Pedersen, 1962; Channon et al., 2000
12. Briskey and Kauffman, 1971
13. Bejerholm, 1984; Sather et al., 1991
14. Fernandez and Tornberg, 1994; Warner, 1997, Channon et al., 2000
15. Eldridge et al., 1993
16. King, 1996)
17. D'Souza, unpublished data
18. LeRoy et al., 1990
19. Estrade et al., 1993
20. Lundström et al., 1998
21. Goransson et al., 1992
22. Channon and Baud, 2000
23. NPPC, 1995
24. MLC, 1992, Table 3
25. D'Souza and Mullan, 2001
26. Hennessy and Wan, 1993
27. Patterson et al., 1990
28. D'Souza et al., 2000
29. Trezona, 2001
30. Cisneros et al., 1996
31. Tarrant, 1998
32. Sather and Jones, 1996
33. Wood, 1984
34. Bailey and Light, 1989
35. Coxon et al., 1986
36. Davies, 1939; Karrick, 1967
37. Hubbard, 1973; Hagiwara et al., 1974
38. Niemack et al., 1979; Kietzman and Jablonski, 1985
39. D'Souza et al., 1998
40. D'Souza et al., 1999a
41. Apple et al., 2000
42. Lawrie, 1998
43. Asghar et al., 1991; Monahan et al., 1992
44. Campbell et al., 1990
45. Dunshea, 1994
46. Weikard et al., 1992
47. Page et al., 1993
48. Barak et al., 1993
49. Cadogan et al., 1993; Henman, 1995; Dunshea and Walton, 1995
50. Dunshea et al., 1998; Thiel et al., 1998
51. Carroll et al., 1999; Dugan et al. 1999; Weigand et al., 2000
52. Dunshea and King, 1994; Dunshea et al., 1993; Dunshea and Walton, 1995
53. Dunshea et al., 1993; Sainz et al., 1993; Xu et al., 1998; Xiao et al., 1999