- Email: [email protected]
Behavioral variation among cloned pigs
Applied Animal Behaviour Science 82 (2003) 151–161
Behavioral variation among cloned pigs夽 Gregory S. Archer a , T.H. Friend a,∗ , J. Piedrahita b,1 , C.H. Nevill a,2 , S. Walker b a b
Department of Animal Science, Texas A&M University, 2471 TAMU, College Station, TX 77843, USA College of Veterinary Medicine, Texas A&M University, 4458 TAMU, College Station, TX 77843, USA
Abstract The variability of behavior among cloned animals has yet to be studied. Through a series of behavior tests, we quantified the variation in food preference, temperament, and time budgets of two genetically identical Duroc litters (n = 5, 4) and their naturally bred controls (n = 4, 4). All litters of pigs were tested for their food preference using apples, bananas, crackers, and carrots. Variation in temperament was determined by timing latency to remove a towel (Towel test) and by counting vocalizations and escape attempts during Back and Pick-up tests. Seventy-two hours of time lapse video were used to determine time budgets of the pigs consisting of the following behaviors: lying in bedding, lying on concrete, standing, feeding, and play/fighting. An F-test was used to determine differences in variation between litter variations. The clones were similarly or more variable (P < 0.05) than the naturally bred controls: in their preference for the foods in 13 of the 16 comparisons; in 5 of the 8 comparisons during the Towel test; in all four comparisons in the Back and Pick-up tests; and in 9 of the 10 comparisons in the time budget analysis. These results reinforce the importance of environmental effects on animal behavior and question the use of cloning by nuclear transfer to replicate animals with specific behavioral characteristics. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Pig; Swine; Clone; Behavior; Variation; Differences
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doi of original article 10.1016/S0168-1591(02)00272-1. Corresponding author. Tel.: +1-979-845-5265; fax: +1-979-845-5292. E-mail address: [email protected] (T.H. Friend). 1 Present address: Department of Anatomy, Physiological Sciences and Radiology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA. 2 Present address: Department of Laboratory Animal Medicine, Southwest Foundation for Biomedical Research, P.O. Box 760549, San Antonio, TX 78245, USA. ∗
0168-1591/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1591(03)00065-0
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1. Introduction It has been demonstrated that the cloning process can result in the birth of normal offspring; however, there are also a considerable number of clones that show a range of abnormalities, including fetal death. Evidence from cloning studies in several mammalian species indicate that epigenetic effects may play a significant role in those abnormalities. It is still not clear which genes are responsible for the abnormalities observed, although imprinted genes are likely candidates (Barlow, 1995; Gold and Pedersen, 1994; Young and Fairburn, 2000). Imprinted genes are implicated in fetal and placental growth, as well as behavior (Murphy et al., 2001; Kobayashi et al., 2002). Thus, it is of interest to determine whether there are behavioral differences between cloned and naturally bred animals. Moreover, cloning by nuclear transfer has been suggested both as a mechanism for replicating animals with specific behavioral characteristics and as an experimental tool to determine genetic versus environmental effects on behavior. In clones, genetic variation is assumed to be eliminated, leaving only environmental stimuli to influence behavioral responses. The use of a multiparous species that allows the generation of litters of clones greatly reduces variation from the maternal environment. By using two groups or litters of clones, we can determine how two groups of genetically identical clones behaved when exposed to a different maternal environment. It is hypothesized that naturally bred litters of pigs have more variability than cloned litters as they have both genetic and environmental variation influencing their behavior, while clones only have environmental variability. We conducted several behavioral tests and established time budgets to analyze this hypothesis.
2. Materials and methods 2.1. Subjects Two litters of cloned female Duroc pigs (Cloned-1 and -2), consisting of five and four pigs, respectively, and two control “litters” of four purebred female Duroc pigs each (Naturally Bred-1 and -2) were used in this study. The cloned litters were born 6 weeks apart, but were from the same fetal cell line and were confirmed to be genetically identical using microsatellite analysis (Walker et al., 2002). The purebred control pigs were of the sasne breed and sex (female), and were born within the same week as their matched cloned litter. The control pigs were obtained from a local Duroc breeder and consisted of a litter of full sibs (Naturally Bred-1) and a litter of half sibs (Naturally Bred-2). Matched litters (Cloned-1 and Naturally Bred-1; Cloned-2 and Naturally Bred-2) started the experiment with average litter weights within 1.5 kg of each other. Cloned-1 average litter weight at the start of the experiment did not include one member of the litter, which was a runt; however, that pig did become comparable to her litter-mates through compensatory gain by 27 weeks of age. All pigs were farrowed in conventional farrowing crates and weaned at 5–6 weeks of age. At weaning, clones and naturally bred controls were placed in adjacent identical pens (1.8 m×6 m) containing a bedded sleeping area, continuous access to a standard commercial ration, and water.
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2.2. Food preference Pigs have a well-developed sense of taste, and owners of pet pigs often find that their pigs have distinct flavor preferences making this an easily tested behavior. Pigs have approximately 850 fungiform papillae on their tongues (Mack et al., 1997), and a total of 15,000 taste buds in their oral and pharyngeal cavities (Kare and Ficken, 1963) that are sensitive to numerous tastes (Danilova et al., 1999). Weanling pigs have preferences for specific flavors of feed (McLaughlin et al., 1983), and certain flavors stimulate the intake of food by weanling pigs (Campbell, 1976; King, 1979). Sweet tastes are preferred by pigs (Kare, 1993; Foster, 1968), and this preference is innate (Kare et al., 1965). Pigs also reject bitter-tasting foods (Kennedy and Baldwin, 1972). All four litters were tested for food preferences at 15–16 weeks (Trial 1) and again at 27 weeks of age (Trial 2). The pigs were offered the following foods by hand: apples, bananas, generic saltine crackers, and carrots. All foods were approximately the same size (3 cm × 3 cm). Lemons and limes were also offered, but were discontinued because none of the pigs would consume them. The pigs willingly approached the experimenter who offered the samples by hand through openings in the panels that formed the sides of the pens. Individual pigs were tested with their group in their home pens. The pigs lined up shoulder to shoulder during these trials. Little aggression was observed as pigs could easily move to a new spot within the group. By testing the pigs in their group, their natural social facilitation was utilized to focus their attention on feeding. To ensure that the pigs actually tasted all types of food, the first offering was placed in each pig’s mouth by the experimenter’s hand. The food type was alternated after each offering until all foods were offered ten times. To ensure that the pig was still hungry and was actually refusing a food, the last food offered was one they had previously accepted, e.g. an apple. 2.3. Temperament evaluation An individual pig’s behavioral responses during non-social tests of temperament is considered to be very consistent (Lawrence et al., 1991). Hence, a “Towel test” and two restraint tests (Back and Pick-up tests) were used to determine variation in temperament of the clone and naturally bred control pigs. 2.3.1. Towel test The Towel test is commonly used to evaluate the temperament of dogs. It was conducted on all four litters between 8 and 9 weeks of age (Trial 1), 14 and 15 weeks of age (Trial 2), and 19 and 21 weeks of age (Trial 3). A towel (406 mm × 610 mm) was placed over the head of each pig extending from behind the ears to over the tip of the nose. Pigs were tested individually in a pen adjacent to their home pen. The time to remove the towel by the pig was recorded; a maximum of 30 s was allowed. This procedure was done 10 consecutive times for each pig during each trial. 2.3.2. Back test The Back test was used by Hessing et al. (1993) to test for individual behavioral characteristics in pigs. This test was conducted only on Cloned-2 and Naturally Bred-2 at 7 weeks
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of age. The other pigs had grown too large to safely place the pigs on their backs. During the Back test, each pig was placed on its dorsal surface and held with one hand on their ventral surface for 1 min. Escape attempts and vocalizations were recorded. A vocalization was defined as a loud squeal and an escape attempts was defined as a struggle to get out of the experimenter’s control. 2.3.3. Pick-up test The Pick-up test involved manually lifting each pig off the ground and supporting the pig with two out-stretched hands for 1 min. Escape attempts and vocalizations were quantified using the same criteria as in the Back test. This test was conducted 20 min after the Back test for both Cloned-2 and Naturally Bred-2 at 7 weeks of age. As with the Back test, the older litters had grown too large to be safely picked up. 2.4. Time budget Additionally, all four litters were time lapse videotaped for a 72 h period between 13 and 15 weeks of age (mean weight 42 kg) to see if quantifiable differences in time budgets existed. During the videotaping, Cloned-1 consisted of four pigs as one was removed for minor veterinary care. These tapes were analyzed using a scan sampling technique at 10 min intervals and the following behaviors were quantified: lying in bedded area, lying on concrete, standing, feeding, and play/fighting. A pig was considered to be feeding if its head was in or above a feeder. It was difficult to differentiate playing from fighting, so both were combined. A pig was considered to be play/fighting when it interacted with another pig in a forceful manner. This consisted of biting, hitting the other pig with its head, or pushing the pig. It also included instances when a group of pigs would run/chase each other (Fraser and Broom, 1996). 2.5. Statistical analysis Although the litters of clones were genetically identical, they were born 6 weeks apart so the tests could not be conducted simultaneously on the two litters of clones and controls. The mean of their response for a particular test could be affected by environmental effects (e.g. weather; different maternal environment for each litter); however, the variance around that mean would be expected to be similar among litters of clones. Hence, variance from the mean is the most important measure in estimating if clones were more or less similar to each other than naturally bred controls. To test this, F-tests were conducted to identify significant differences (P < 0.05) in variances between matched litters (Cloned-1 versus Naturally Bred-1 and Cloned-2 versus Naturally Bred-2) and within cloned and naturally bred control populations (Cloned-1 versus Cloned-2 and Naturally Bred-1 versus Naturally Bred-2). The matched litter comparisons will largely investigate the epigenetic influences on litter variations while the within population comparisons will investigate both epigenetic and environmental influences on litter variations. A sign test was also conducted to determine if there was a disproportionate amount of times that either the cloned litters or naturally bred litters were found to be significantly more variable by the F-tests.
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Fig. 1. Variation in food preferences between two litters of cloned and naturally bred groups of pigs. Bars within trials with the same letter are significantly different (P < 0.05).
3. Results and discussions 3.1. Variation of food preference The food preference test found differences in variation between and within cloned and naturally bred animals. Most of the pigs in the study ate all the apples, but in Cloned-1, one pig refused all the apples and another refused two apples during the first apple trial (Fig. 1). During the second apple trial, however, all litters demonstrated equal apple preference by consuming all offerings indicating that the initial food preference had disappeared. Preference for bananas varied in both trials for both litters of clones, while only Naturally Bred-1 showed variation (Fig. 1). In the first banana trial, clones were equally or more variable than their matched litters. In the second banana trial, both litters of clones were more variable than their matched litters. In the first cracker trial, Cloned-1 was more variable than Cloned-2 (Fig. 1). In the second trial, Naturally Bred-1 was less variable than Cloned-1 and Naturally Bred-2 (Fig. 1) because all Naturally Bred-1 members ate all crackers offered. Cloned-2 was also less variable than Cloned-1 and Naturally Bred-2 in the second cracker trial. In the first trial evaluating preference for carrots, Cloned-1 was more variable than Cloned-2 and Naturally Bred-2 was less variable than Cloned-2 (Fig. 1). In the second
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Fig. 2. Individual litter variation in responses during the Towel test. Bars within trials with the same letter are significantly different in variation (P < 0.05). Results for Towel Trials 1–3 were averaged for each individual in each litter and the variability of those means is shown as “Towel mean”.
trial, Cloned-1 and Naturally Bred-2 were more variable than the other two litters in their preference for carrots. Overall, the test indicated that variability in taste preference in cloned animals can be as great or greater than in naturally bred animals. 3.2. Temperament evaluation 3.2.1. Variation of Towel test In these trials, there were differences in the variances of matched litters and within cloned and naturally bred populations (Fig. 2). In the first trial, cloned litters were different from their matched control litters in opposite directions and the naturally bred litters were different from each other. In the second trial, Naturally Bred-1 and Cloned-2 were more variable than Naturally Bred-2. In the third trial, Naturally Bred-1 was more variable than Cloned-1 and Naturally Bred-2 (Fig. 2), which was the case in Trial 1, suggesting that in this particular behavior initial differences are persistent. When the results of all three trials were averaged for each pig and the overall variability was calculated for each
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Fig. 3. Individual litter variation in responses during the Back and Pick-up tests. There were no differences in variation (P > 0.05).
litter, Naturally Bred-1 was still significantly more variable than Cloned-1 and Naturally Bred-2. 3.2.2. Variation of back and Pick-up tests During the Back and Pick-up trials, there were no significant differences between the variance of cloned and naturally bred animals in the number of vocalizations or in the number of escape attempts (Fig. 3). Although no significant differences were observed, in the Back test, the cloned litter had a variance of 47 and the naturally bred litter had a variance of 12.9. Interestingly, this trend was reversed in the Pick-up test where the naturally bred litter had a variance of 22.3 and the cloned litter had a variance of 4.7. 3.3. Variation of time budget The two cloned litters differed from each other in variation of time spent lying on concrete. Also, Cloned-1 and Naturally Bred-1 were different in variation of time spent feeding (Fig. 4). Cloned-1 was also less variable than Cloned-2 in this behavior. The last difference in time budget variation existed between the two naturally bred litters for play/fighting behavior (Fig. 4).
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Fig. 4. Individual litter variation in behavior for the time budget. Bars within behaviors with the same letter are significantly different in variation (P < 0.05).
3.4. Sign test results The F-test found that the cloned litters had greater variation 9 times, the naturally bred litters had greater variation 7 times, and the litters had similar variation 22 times. The sign test indicated that this difference between the cloned and naturally bred litters was not significant (P = 0.80), further demonstrating that the cloned and naturally bred litters were equally variable in the F-tests. This negates the hypothesis that cloned litters are significantly less variable in the traits studied more than the naturally bred litters. 3.5. Means of all responses Table 1 was included for the reference of the readers. In interpreting this table, however, it is important to remember that it is the variance around these means, and not the means themselves, that are the focus of this study. Possible environmental differences arising from the litters being born 6 weeks apart, having different maternal environments, etc. along with the low number of animals per litter, makes statistical analysis of the means problematic. Also, conducting an F-test on the variance was the most appropriate method of analyzing these data.
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Table 1 Mean response per litter in the food preference, towel, and restraint tests, and distribution of time performing behaviors in the time budget Cloned-1
Naturally Bred-1
Cloned-2
Naturally Bred-2
7.6 ± 4.3 10.0 ± 0.0
10.0 ± 0.0 10.0 ± 0.0
9.8 ± 0.5 10.0 ± 0.0
10.0 ± 0.0 10.0 ± 0.0
Banana Trial 1 Trial 2
8.2 ± 4.0 6.4 ± 5.0
8.0 ± 4.0 10.0 ± 0.0
1.5 ± 2.4 1.5 ± 3.0
10.0 ± 0.0 10.0 ± 0.0
Cracker Trial 1 Trial 2
6.2 ± 5.2 9.0 ± 2.2
8.5 ± 3.0 10 0 ± 0.0
9.5 + 1.0 9.8 ± 0.5
8.3 ± 1.7 8.1 + 3.0
Carrot Trial 1 Trial 2
4.8 ± 4.3 8.2 ± 2.5
3.5 ± 4.7 10.0 ± 0.0
9.3 ± 1.0 9.0 ± 0.8
7.0 ± 3.5 5.3 ± 5.5
2.2 ± 1.1 3.3 + 0.9 6.5 ± 2.2 4.0 ± 1.1
2.5 ± 4.0 1.5 ± 0.9 10.7 ± 7.4 4.9 ± 3.0
2.9 + 1.4 0.3 ± 0.5 3.4 ± 0.7 2.2 ± 0.6
0.0 + 0.00 0.2 ± 0.1 3.4 ± 1.8 1.2 ± 0.6
15.0 ± 2.2 3.5 ± 1.0 10.5 ± 6.9 2.8 ± 3.6
6.5 ± 4.7 2.3 ± 1.7 4.8 ± 3.6 2.0 ± 1.6
76.8 ± 3.1 6.2 ± 2.2 8.4 ± 0.8 7.7 ± 2.1 0.9 ± 0.6
76.8 ± 3.6 3.8 ± 1.1 10.4 ± 1.5 7.7 ± 1.6 1.3 ± 0.6
Food preference Apple Trial 1 Trial 2
testa
Towel testb Towel Trial 1 Trial 2 Trial 3 Mean Restraint testsc Pick-up vocalization Pick-up escape attempts Back test vocalization Back test escape attempts Time budgetd Lying in bedding area Lying on concrete Standing Feeding Play/fighting
– – – – 78.1 ± 1.6 10.9 ± 0.5 6.6 ± 1.3 4.0 ± 0.5 0.4 ± 0.3
– – – – 83.3 ± 2.9 5.6 ± 0.8 4.6 ± 0.8 6.4 ± 1.8 0.1 ± 0.1
Mean number eaten ± S.D. Mean latency (s) to remover towel ± S.D. c Mean number of occurrences in 1 min ± S.D. d Mean (%) of observations per 24 h ± S.D. a
b
3.6. Informal observations Differences in personality among the clones were also apparent through informal observations. Some individual clones were more aggressive, while some were “friendlier” to their litter-mates. Some members of cloned litters also consistently kept their bedded sleeping area clean, while other pigs would selectively urinate or defecate in the sleeping area. Certain members of the cloned litters also appeared to participate in belly nosing more
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than other members of their litter. Unfortunately, urination and defecation patterns, and belly nosing could not be accurately quantified from the collected video. There was also considerable variation in all litters in which individuals approached the researcher.
4. Conclusion From the tests conducted, it appears that litters of genetically identical clones are not any more consistent in their behavior than litters of naturally bred animals. This reinforces the importance of uterine effects, maternal behavior, or environmental effects (e.g. weather) in affecting behavior. Factors such as mitochondrial background or methylation-associated variability introduced during the cloning process may also contribute to the variation of behavior among cloned animals. It appears from these observations that the goal of using nuclear transfer to replicate animals to reproduce certain behavioral characteristics is an unrealistic expectation.
Acknowledgements We thank Dr. Omer Jenkins for his assistance with the statistical analysis.
References Barlow, D.P., 1995. Genetic imprinting in mammals. Science 270, 1610–1613. Campbell, R.G., 1976. A note on the use of a feed flavour to stimulate the feed intake of weanling pigs. Anim. Prod. 23, 417–419. Danilova, V., Roberts, R., Hellekant, G., 1999. Responses of single taste fibers and whole Chorda tympani and Glossopharyngeal Nerve in domestic pig. Sus. Scrofa. Chem. Senses. 24, 301–316. Fraser, A.F., Broom, D.M., 1996. Farm Animal Behavior and Welfare. Bailliere Tindall, London, pp. 251–252. Foster, R., 1968. The reward value of saccharin solution prior to eating experience. Psychonomic Sci. 10, 83–84. Gold, J.D., Pedersen, R.A., 1994. Mechanisms of genomic imprinting in mammals. Curr. Top. Dev. Biol. 29, 227–280. Hessing, M.J.C., Hagelso, A.M., van Beek, J.A.M., Wiepkema, P.R., Schouten, W.G.P., Krukow, R., 1993. Individual behavioural characteristics in pigs. Appl. Anim. Behav. Sci. 37, 285–295. Kare, M.R., 1993. Dukes’ Physiology of Domestic Animals. 11th ed. Comstock Publishing, New York, pp. 816–835. Kare, M.R., Ficken, M.S., 1963. Comparative studies on the sense of taste. In: Proceedings of the First International Symposium on Olfaction and Taste. Stockholm, pp. 285–297. Kare, M.R., Pond, W.G., Campbell, J., 1965. Observations on the taste reactions in pigs. Anim. Behav. 13, 265–269. Kennedy, J.M., Baldwin, B.A., 1972. Taste preferences in pigs for nutritive and non-nutritive sweet solutions. Anim. Behav. 20, 706–718. King, R.H., 1979. The effect of adding a feed flavour to the diets of young pigs before and after weaning. Aust. J. Agric. Anim. Husb. 19, 695–697. Kobayashi, S., Kohda, T., Ichikawa, H., Ogura, A., Ohki, M., Kaneko-Ishino, T., Ishino, F., 2002. Paternal expression of a novel imprinted gene, Peg12/Frat3, in the Mouse 7C region homologous to the Prader–Willi syndrome region. Biochem. Biophys. Res. Commun. 290, 403–408. Lawrence, A.B., Terlouw, E.M.C., Illius, A.W., 1991. Individual differences in behavioral responses of pigs exposed to non-social and social challenges. Appl. Anim. Behav. Sci. 30, 73–86.
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Mack, A., Singh, A., Gilroy, C., Ireland, W., 1997. Porcine lingual taste buds: a quantitative study. Anal. Rec. 247, 33–37. McLaughlin, C.L., Baile, C.A., Buckholtz, L.L., Freeman, S.K., 1983. Preferred flavors and performance of weanling pigs. J. Anim. Sci. 56, 1287–1293. Murphy, S.K., Wylie, A.A., Jirtle, R.L., 2001. Imprinting of PEG3, the human homologue of a mouse gene involved in nurturing behavior. Genomics 71, 110–117. Walker, S., Shin, T.Y., Zaunbrecher, G.M., Romano, J.E., Johnson, G.A., Bazer, F.W., Piedralihita, J.A., 2002. A highly efficient method for porcine cloning by nuclear transfer using in vitro-mature oocytes. Clon. Stem Cells 4 (2), 105–112. Young, L.E., Fairburn, H.R., 2000. Improving the safety of embryo technologies: possible role of genotnic imprinting. Theriogenology 53, 448–627.
Behavioral variation among cloned pigs夽 Gregory S. Archer a , T.H. Friend a,∗ , J. Piedrahita b,1 , C.H. Nevill a,2 , S. Walker b a b
Department of Animal Science, Texas A&M University, 2471 TAMU, College Station, TX 77843, USA College of Veterinary Medicine, Texas A&M University, 4458 TAMU, College Station, TX 77843, USA
Abstract The variability of behavior among cloned animals has yet to be studied. Through a series of behavior tests, we quantified the variation in food preference, temperament, and time budgets of two genetically identical Duroc litters (n = 5, 4) and their naturally bred controls (n = 4, 4). All litters of pigs were tested for their food preference using apples, bananas, crackers, and carrots. Variation in temperament was determined by timing latency to remove a towel (Towel test) and by counting vocalizations and escape attempts during Back and Pick-up tests. Seventy-two hours of time lapse video were used to determine time budgets of the pigs consisting of the following behaviors: lying in bedding, lying on concrete, standing, feeding, and play/fighting. An F-test was used to determine differences in variation between litter variations. The clones were similarly or more variable (P < 0.05) than the naturally bred controls: in their preference for the foods in 13 of the 16 comparisons; in 5 of the 8 comparisons during the Towel test; in all four comparisons in the Back and Pick-up tests; and in 9 of the 10 comparisons in the time budget analysis. These results reinforce the importance of environmental effects on animal behavior and question the use of cloning by nuclear transfer to replicate animals with specific behavioral characteristics. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Pig; Swine; Clone; Behavior; Variation; Differences
夽
doi of original article 10.1016/S0168-1591(02)00272-1. Corresponding author. Tel.: +1-979-845-5265; fax: +1-979-845-5292. E-mail address: [email protected] (T.H. Friend). 1 Present address: Department of Anatomy, Physiological Sciences and Radiology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA. 2 Present address: Department of Laboratory Animal Medicine, Southwest Foundation for Biomedical Research, P.O. Box 760549, San Antonio, TX 78245, USA. ∗
0168-1591/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1591(03)00065-0
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1. Introduction It has been demonstrated that the cloning process can result in the birth of normal offspring; however, there are also a considerable number of clones that show a range of abnormalities, including fetal death. Evidence from cloning studies in several mammalian species indicate that epigenetic effects may play a significant role in those abnormalities. It is still not clear which genes are responsible for the abnormalities observed, although imprinted genes are likely candidates (Barlow, 1995; Gold and Pedersen, 1994; Young and Fairburn, 2000). Imprinted genes are implicated in fetal and placental growth, as well as behavior (Murphy et al., 2001; Kobayashi et al., 2002). Thus, it is of interest to determine whether there are behavioral differences between cloned and naturally bred animals. Moreover, cloning by nuclear transfer has been suggested both as a mechanism for replicating animals with specific behavioral characteristics and as an experimental tool to determine genetic versus environmental effects on behavior. In clones, genetic variation is assumed to be eliminated, leaving only environmental stimuli to influence behavioral responses. The use of a multiparous species that allows the generation of litters of clones greatly reduces variation from the maternal environment. By using two groups or litters of clones, we can determine how two groups of genetically identical clones behaved when exposed to a different maternal environment. It is hypothesized that naturally bred litters of pigs have more variability than cloned litters as they have both genetic and environmental variation influencing their behavior, while clones only have environmental variability. We conducted several behavioral tests and established time budgets to analyze this hypothesis.
2. Materials and methods 2.1. Subjects Two litters of cloned female Duroc pigs (Cloned-1 and -2), consisting of five and four pigs, respectively, and two control “litters” of four purebred female Duroc pigs each (Naturally Bred-1 and -2) were used in this study. The cloned litters were born 6 weeks apart, but were from the same fetal cell line and were confirmed to be genetically identical using microsatellite analysis (Walker et al., 2002). The purebred control pigs were of the sasne breed and sex (female), and were born within the same week as their matched cloned litter. The control pigs were obtained from a local Duroc breeder and consisted of a litter of full sibs (Naturally Bred-1) and a litter of half sibs (Naturally Bred-2). Matched litters (Cloned-1 and Naturally Bred-1; Cloned-2 and Naturally Bred-2) started the experiment with average litter weights within 1.5 kg of each other. Cloned-1 average litter weight at the start of the experiment did not include one member of the litter, which was a runt; however, that pig did become comparable to her litter-mates through compensatory gain by 27 weeks of age. All pigs were farrowed in conventional farrowing crates and weaned at 5–6 weeks of age. At weaning, clones and naturally bred controls were placed in adjacent identical pens (1.8 m×6 m) containing a bedded sleeping area, continuous access to a standard commercial ration, and water.
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2.2. Food preference Pigs have a well-developed sense of taste, and owners of pet pigs often find that their pigs have distinct flavor preferences making this an easily tested behavior. Pigs have approximately 850 fungiform papillae on their tongues (Mack et al., 1997), and a total of 15,000 taste buds in their oral and pharyngeal cavities (Kare and Ficken, 1963) that are sensitive to numerous tastes (Danilova et al., 1999). Weanling pigs have preferences for specific flavors of feed (McLaughlin et al., 1983), and certain flavors stimulate the intake of food by weanling pigs (Campbell, 1976; King, 1979). Sweet tastes are preferred by pigs (Kare, 1993; Foster, 1968), and this preference is innate (Kare et al., 1965). Pigs also reject bitter-tasting foods (Kennedy and Baldwin, 1972). All four litters were tested for food preferences at 15–16 weeks (Trial 1) and again at 27 weeks of age (Trial 2). The pigs were offered the following foods by hand: apples, bananas, generic saltine crackers, and carrots. All foods were approximately the same size (3 cm × 3 cm). Lemons and limes were also offered, but were discontinued because none of the pigs would consume them. The pigs willingly approached the experimenter who offered the samples by hand through openings in the panels that formed the sides of the pens. Individual pigs were tested with their group in their home pens. The pigs lined up shoulder to shoulder during these trials. Little aggression was observed as pigs could easily move to a new spot within the group. By testing the pigs in their group, their natural social facilitation was utilized to focus their attention on feeding. To ensure that the pigs actually tasted all types of food, the first offering was placed in each pig’s mouth by the experimenter’s hand. The food type was alternated after each offering until all foods were offered ten times. To ensure that the pig was still hungry and was actually refusing a food, the last food offered was one they had previously accepted, e.g. an apple. 2.3. Temperament evaluation An individual pig’s behavioral responses during non-social tests of temperament is considered to be very consistent (Lawrence et al., 1991). Hence, a “Towel test” and two restraint tests (Back and Pick-up tests) were used to determine variation in temperament of the clone and naturally bred control pigs. 2.3.1. Towel test The Towel test is commonly used to evaluate the temperament of dogs. It was conducted on all four litters between 8 and 9 weeks of age (Trial 1), 14 and 15 weeks of age (Trial 2), and 19 and 21 weeks of age (Trial 3). A towel (406 mm × 610 mm) was placed over the head of each pig extending from behind the ears to over the tip of the nose. Pigs were tested individually in a pen adjacent to their home pen. The time to remove the towel by the pig was recorded; a maximum of 30 s was allowed. This procedure was done 10 consecutive times for each pig during each trial. 2.3.2. Back test The Back test was used by Hessing et al. (1993) to test for individual behavioral characteristics in pigs. This test was conducted only on Cloned-2 and Naturally Bred-2 at 7 weeks
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of age. The other pigs had grown too large to safely place the pigs on their backs. During the Back test, each pig was placed on its dorsal surface and held with one hand on their ventral surface for 1 min. Escape attempts and vocalizations were recorded. A vocalization was defined as a loud squeal and an escape attempts was defined as a struggle to get out of the experimenter’s control. 2.3.3. Pick-up test The Pick-up test involved manually lifting each pig off the ground and supporting the pig with two out-stretched hands for 1 min. Escape attempts and vocalizations were quantified using the same criteria as in the Back test. This test was conducted 20 min after the Back test for both Cloned-2 and Naturally Bred-2 at 7 weeks of age. As with the Back test, the older litters had grown too large to be safely picked up. 2.4. Time budget Additionally, all four litters were time lapse videotaped for a 72 h period between 13 and 15 weeks of age (mean weight 42 kg) to see if quantifiable differences in time budgets existed. During the videotaping, Cloned-1 consisted of four pigs as one was removed for minor veterinary care. These tapes were analyzed using a scan sampling technique at 10 min intervals and the following behaviors were quantified: lying in bedded area, lying on concrete, standing, feeding, and play/fighting. A pig was considered to be feeding if its head was in or above a feeder. It was difficult to differentiate playing from fighting, so both were combined. A pig was considered to be play/fighting when it interacted with another pig in a forceful manner. This consisted of biting, hitting the other pig with its head, or pushing the pig. It also included instances when a group of pigs would run/chase each other (Fraser and Broom, 1996). 2.5. Statistical analysis Although the litters of clones were genetically identical, they were born 6 weeks apart so the tests could not be conducted simultaneously on the two litters of clones and controls. The mean of their response for a particular test could be affected by environmental effects (e.g. weather; different maternal environment for each litter); however, the variance around that mean would be expected to be similar among litters of clones. Hence, variance from the mean is the most important measure in estimating if clones were more or less similar to each other than naturally bred controls. To test this, F-tests were conducted to identify significant differences (P < 0.05) in variances between matched litters (Cloned-1 versus Naturally Bred-1 and Cloned-2 versus Naturally Bred-2) and within cloned and naturally bred control populations (Cloned-1 versus Cloned-2 and Naturally Bred-1 versus Naturally Bred-2). The matched litter comparisons will largely investigate the epigenetic influences on litter variations while the within population comparisons will investigate both epigenetic and environmental influences on litter variations. A sign test was also conducted to determine if there was a disproportionate amount of times that either the cloned litters or naturally bred litters were found to be significantly more variable by the F-tests.
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Fig. 1. Variation in food preferences between two litters of cloned and naturally bred groups of pigs. Bars within trials with the same letter are significantly different (P < 0.05).
3. Results and discussions 3.1. Variation of food preference The food preference test found differences in variation between and within cloned and naturally bred animals. Most of the pigs in the study ate all the apples, but in Cloned-1, one pig refused all the apples and another refused two apples during the first apple trial (Fig. 1). During the second apple trial, however, all litters demonstrated equal apple preference by consuming all offerings indicating that the initial food preference had disappeared. Preference for bananas varied in both trials for both litters of clones, while only Naturally Bred-1 showed variation (Fig. 1). In the first banana trial, clones were equally or more variable than their matched litters. In the second banana trial, both litters of clones were more variable than their matched litters. In the first cracker trial, Cloned-1 was more variable than Cloned-2 (Fig. 1). In the second trial, Naturally Bred-1 was less variable than Cloned-1 and Naturally Bred-2 (Fig. 1) because all Naturally Bred-1 members ate all crackers offered. Cloned-2 was also less variable than Cloned-1 and Naturally Bred-2 in the second cracker trial. In the first trial evaluating preference for carrots, Cloned-1 was more variable than Cloned-2 and Naturally Bred-2 was less variable than Cloned-2 (Fig. 1). In the second
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Fig. 2. Individual litter variation in responses during the Towel test. Bars within trials with the same letter are significantly different in variation (P < 0.05). Results for Towel Trials 1–3 were averaged for each individual in each litter and the variability of those means is shown as “Towel mean”.
trial, Cloned-1 and Naturally Bred-2 were more variable than the other two litters in their preference for carrots. Overall, the test indicated that variability in taste preference in cloned animals can be as great or greater than in naturally bred animals. 3.2. Temperament evaluation 3.2.1. Variation of Towel test In these trials, there were differences in the variances of matched litters and within cloned and naturally bred populations (Fig. 2). In the first trial, cloned litters were different from their matched control litters in opposite directions and the naturally bred litters were different from each other. In the second trial, Naturally Bred-1 and Cloned-2 were more variable than Naturally Bred-2. In the third trial, Naturally Bred-1 was more variable than Cloned-1 and Naturally Bred-2 (Fig. 2), which was the case in Trial 1, suggesting that in this particular behavior initial differences are persistent. When the results of all three trials were averaged for each pig and the overall variability was calculated for each
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Fig. 3. Individual litter variation in responses during the Back and Pick-up tests. There were no differences in variation (P > 0.05).
litter, Naturally Bred-1 was still significantly more variable than Cloned-1 and Naturally Bred-2. 3.2.2. Variation of back and Pick-up tests During the Back and Pick-up trials, there were no significant differences between the variance of cloned and naturally bred animals in the number of vocalizations or in the number of escape attempts (Fig. 3). Although no significant differences were observed, in the Back test, the cloned litter had a variance of 47 and the naturally bred litter had a variance of 12.9. Interestingly, this trend was reversed in the Pick-up test where the naturally bred litter had a variance of 22.3 and the cloned litter had a variance of 4.7. 3.3. Variation of time budget The two cloned litters differed from each other in variation of time spent lying on concrete. Also, Cloned-1 and Naturally Bred-1 were different in variation of time spent feeding (Fig. 4). Cloned-1 was also less variable than Cloned-2 in this behavior. The last difference in time budget variation existed between the two naturally bred litters for play/fighting behavior (Fig. 4).
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Fig. 4. Individual litter variation in behavior for the time budget. Bars within behaviors with the same letter are significantly different in variation (P < 0.05).
3.4. Sign test results The F-test found that the cloned litters had greater variation 9 times, the naturally bred litters had greater variation 7 times, and the litters had similar variation 22 times. The sign test indicated that this difference between the cloned and naturally bred litters was not significant (P = 0.80), further demonstrating that the cloned and naturally bred litters were equally variable in the F-tests. This negates the hypothesis that cloned litters are significantly less variable in the traits studied more than the naturally bred litters. 3.5. Means of all responses Table 1 was included for the reference of the readers. In interpreting this table, however, it is important to remember that it is the variance around these means, and not the means themselves, that are the focus of this study. Possible environmental differences arising from the litters being born 6 weeks apart, having different maternal environments, etc. along with the low number of animals per litter, makes statistical analysis of the means problematic. Also, conducting an F-test on the variance was the most appropriate method of analyzing these data.
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Table 1 Mean response per litter in the food preference, towel, and restraint tests, and distribution of time performing behaviors in the time budget Cloned-1
Naturally Bred-1
Cloned-2
Naturally Bred-2
7.6 ± 4.3 10.0 ± 0.0
10.0 ± 0.0 10.0 ± 0.0
9.8 ± 0.5 10.0 ± 0.0
10.0 ± 0.0 10.0 ± 0.0
Banana Trial 1 Trial 2
8.2 ± 4.0 6.4 ± 5.0
8.0 ± 4.0 10.0 ± 0.0
1.5 ± 2.4 1.5 ± 3.0
10.0 ± 0.0 10.0 ± 0.0
Cracker Trial 1 Trial 2
6.2 ± 5.2 9.0 ± 2.2
8.5 ± 3.0 10 0 ± 0.0
9.5 + 1.0 9.8 ± 0.5
8.3 ± 1.7 8.1 + 3.0
Carrot Trial 1 Trial 2
4.8 ± 4.3 8.2 ± 2.5
3.5 ± 4.7 10.0 ± 0.0
9.3 ± 1.0 9.0 ± 0.8
7.0 ± 3.5 5.3 ± 5.5
2.2 ± 1.1 3.3 + 0.9 6.5 ± 2.2 4.0 ± 1.1
2.5 ± 4.0 1.5 ± 0.9 10.7 ± 7.4 4.9 ± 3.0
2.9 + 1.4 0.3 ± 0.5 3.4 ± 0.7 2.2 ± 0.6
0.0 + 0.00 0.2 ± 0.1 3.4 ± 1.8 1.2 ± 0.6
15.0 ± 2.2 3.5 ± 1.0 10.5 ± 6.9 2.8 ± 3.6
6.5 ± 4.7 2.3 ± 1.7 4.8 ± 3.6 2.0 ± 1.6
76.8 ± 3.1 6.2 ± 2.2 8.4 ± 0.8 7.7 ± 2.1 0.9 ± 0.6
76.8 ± 3.6 3.8 ± 1.1 10.4 ± 1.5 7.7 ± 1.6 1.3 ± 0.6
Food preference Apple Trial 1 Trial 2
testa
Towel testb Towel Trial 1 Trial 2 Trial 3 Mean Restraint testsc Pick-up vocalization Pick-up escape attempts Back test vocalization Back test escape attempts Time budgetd Lying in bedding area Lying on concrete Standing Feeding Play/fighting
– – – – 78.1 ± 1.6 10.9 ± 0.5 6.6 ± 1.3 4.0 ± 0.5 0.4 ± 0.3
– – – – 83.3 ± 2.9 5.6 ± 0.8 4.6 ± 0.8 6.4 ± 1.8 0.1 ± 0.1
Mean number eaten ± S.D. Mean latency (s) to remover towel ± S.D. c Mean number of occurrences in 1 min ± S.D. d Mean (%) of observations per 24 h ± S.D. a
b
3.6. Informal observations Differences in personality among the clones were also apparent through informal observations. Some individual clones were more aggressive, while some were “friendlier” to their litter-mates. Some members of cloned litters also consistently kept their bedded sleeping area clean, while other pigs would selectively urinate or defecate in the sleeping area. Certain members of the cloned litters also appeared to participate in belly nosing more
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than other members of their litter. Unfortunately, urination and defecation patterns, and belly nosing could not be accurately quantified from the collected video. There was also considerable variation in all litters in which individuals approached the researcher.
4. Conclusion From the tests conducted, it appears that litters of genetically identical clones are not any more consistent in their behavior than litters of naturally bred animals. This reinforces the importance of uterine effects, maternal behavior, or environmental effects (e.g. weather) in affecting behavior. Factors such as mitochondrial background or methylation-associated variability introduced during the cloning process may also contribute to the variation of behavior among cloned animals. It appears from these observations that the goal of using nuclear transfer to replicate animals to reproduce certain behavioral characteristics is an unrealistic expectation.
Acknowledgements We thank Dr. Omer Jenkins for his assistance with the statistical analysis.
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