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Abstract

Research on the impact of bioactive compounds on the development and functional maturation of the gastrointestinal (GI) tract using newborn and juvenile rats has greatly contributed to the knowledge of GI physiology and to the improved clinical management of both premature and full-term newborns. Of the animal models available, two types have been described for use with young rats – maintenance models and substitution models. Maintenance models are those in which the young are reared with the dam and therefore benefit from continuation of natural nutrition and maternal care. Substitution models are those in which the young are reared in the absence of the dam using artificially formulated milk delivered by various means into specific GI sites. In this review, we describe these models and their operation, and discuss the strengths and weaknesses of each. Attention is also given to questions of scientific validity and some animal welfare issues raised by the use of these models.

Keywords

Artificial rearing pup in a cup rat pup gastrointestinal development animal welfare

1. Introduction

Numerous studies have contributed to the understanding of the mechanisms that are responsible for the development of the mammalian digestive system (Henning 1986, Montgomery et al. 1999, Walthall et al. 2005), i.e. its progression from a stage of immaturity to that of maturity. The digestive system begins to develop in utero such that pre-emptive development of its form and function prepares the neonate for the extrauterine environment (Henning 1986, Walthall et al. 2005) and subsequent adulthood. Early postnatal development, particularly that of the gastrointestinal tract (GI), is cued by bioactive elements in the colostrum and milk (Sheard & Walker 1988, Koldovsky 1989, Xu 1996). The roles of these ingested bioactives have been explored with a range of models based on various species, including the rat and mouse.

Preweanling rats are particularly suitable for bioactive studies because of their availability, ease of handling, accessibility during different stages of development and well-described physiology. The available models may be categorized into those in which the young are reared by the dam (maintenance models) and those where the young are reared in the absence of the dam (substitution models), with or without the presence of their siblings.

The use of these models raises issues of scientific validity as well as those of animal welfare. Maintenance models, in relying on the provision of milk by the dam, generally cannot be used to explore the effects of factor deficiency, and must rely either on premature delivery or supraphysiological or pharmacological dosage with one or several factors to produce recordable effects. Their principal advantage lies in the avoidance of invasive surgical procedures and in the continuation of normal maternal care, which together reduce the likelihood of pathological sequelae and avoid a number of animal welfare problems associated with substitution models. The principal scientific disadvantage of maintenance models, which has led to the use of substitution models, is the likelihood of saturation effects in the dose-response curves of bioactive agents, so that augmentation of the normal maternal dosage via her milk produces no additional effects.

Substitution models can be used to investigate the effects of formulated milk that have particular bioactive deficiencies. The principal problem with the use of such a strategy is the limited knowledge of normal patterns of variation of the nutritional and hormonal components of maternal milk. Again, removal of the pup from maternal care requires that intake be maintained by other means. Comparisons of the mortality and morbidity of maternally-reared pups with those used in substitution models raise questions about the adequacy of such neonatal maintenance. At the limit case, substitution models may assess the ability of particular milk-borne factors to rescue the pup from conditions resulting from an inadequate maintenance procedure rather than assessing their role in normal development. While it may be envisaged that this scenario would be of benefit in identifying bioactives that enhance recovery from deficient states, it may be difficult to distinguish physiological from patho-physiological effects.

In this review, we will summarize the procedures used in the range of maintenance and substitution models that are in current use, provide a discussion of the strengths and weaknesses of the models and consider what models may be appropriate for particular investigations. Finally, we will discuss a number of scientific and animal welfare issues that arise from the use of these models.

2. Materials and methods

2.1 Maintenance models

In maintenance models, preweanling rats are kept with and reared by the dam. The young are intermittently detached from the maternal nipple and supplemented with selected bioactive substances using a variety of techniques to deliver them into the mouth, oesophagus or stomach.

2.1.1 Supplementation techniques

2.1.1.1 Oral delivery

Simple protocols using eyedroppers or pipette tips attached to syringes have (anecdotally) been used to deliver fluids into the oral cavity. Customized bottles and teats have also been developed for use with mouse and rat pups (Hoshiba 2004, Lim et al. 2005).

2.1.1.2 Oesophageal gavage

A ball-tipped stainless steel or plastic needle or a length of plastic tubing is introduced into the mouth and advanced down the oesophagus as the conscious animal swallows (Smith & Kelleher 1973, Waynforth & Flecknell 1992, Watanabe et al. 2003). When the tip of the device is judged to be in the lower oesophagus or at the entrance to the stomach, the contents of the attached syringe are delivered at the required rate and the device is subsequently withdrawn. While gavage has greater potential for trauma than oral delivery, it allows a precise volume of fluid to be administered in a dose that is appropriately adjusted for body mass with a lower possibility of spillage.

2.1.2 Strengths

The strength of maintenance models lies in maternal care by the dam. Thus, the dam provides for the pups' nutritional requirements, as well as aiding its thermal regulation, eliciting urination and defaecation with appropriate tactile stimulation, and giving the somatosensory stimulation necessary for normal neurological development (Rosenblatt & Lehrman 1963). Social interactions with siblings may also contribute to the pups' wellbeing and enhance behavioural development (Alberts 1978, Uvnas-Moberg 1997, Schank & Alberts 2000). Newborn and juvenile rats that are reared with the dam develop a repertoire of maternally directed seeking and orientating behaviours (Polan & Hofer 1999), e.g. suckling, which subsequently serve to augment normal development and assist in the transition to adult behaviours (Hall 1990, Smith 2006). Intermittent oral dosing with bioactives allows these behaviours to develop and continue with minimal disturbance.

2.1.3 Problems

The limitation of maintenance models regarding augmentation of dosage with bioactives and the consequent difficulty of distinguishing pharmacological from physiological effects has been mentioned above. However, the prospect of cross-fostering onto gene-silenced (i.e. ‘knockout’) rats and mice (Melton 1994, Bockamp et al. 2002, Zan et al. 2003) may soon permit exploration of specific deficiencies in a maintenance model.

A number of factors may confound the use of conventional maintenance models. It is generally assumed that appropriate nourishment of the mother will maintain production of milk of a composition suitable for optimum development (Lau & Simpson 2004). Although maternal nutrient reserves may reduce deleterious effects of a nutritional deficit (Rasmussen 1998), manipulation of the maternal diet may alter milk composition (Muller & Cox 1946, Forbes et al. 1977, Rasmussen 1998). Thus, ongoing analysis of maternal milk is necessary, particularly in situations when the effects of nutrient supplementation are being investigated. Similarly, the concentrations of bioactives in maternal milk should be evaluated when bioactives are being supplemented.

Depleted maternal nutritional status may adversely influence milk composition and production and confound the effects of a bioactive supplement on pup development by adversely affecting body growth (Forbes et al. 1977, Rasmussen & Warman 1983, Del Prado et al. 1997, Weaver et al. 1998, Matias et al. 2003), the development of the GI tract (Weaver et al. 1998) and endocrine signalling, e.g. corticosterone concentrations (Kliewer & Rasmussen 1987) and leptin concentrations (Korotkova et al. 2001). Reduction of maternal milk production may also result from reduced maternal food intake (Rasmussen 1998), or from a reduction in mammary stimulation and/or milk demand when pups are repeatedly removed from the mother. Intermittent removal may also lead to rejection of a pup or litter (Libbin & Person 1979) or to cannibalism by the dam, but such occurrences are rare, provided appropriate precautions are taken. Stress-induced increases in corticosterone concentrations following short periods of isolation and handling are known to be short-lived (Stanton et al. 1988, Cirulli et al. 1992). Also, brief periods of physical separation of the order of 1 h may not affect growth rate of newborn or juvenile rats even when the periods of separation are repeated over several days (Kehoe et al. 1996).

The restraints necessary for oral delivery and gavage may induce stress, or cause physical damage to pups. Again, inaccurate intraoral placement of the tip of the device may cause spillage rendering bioactive dosage inconstant and increase the likelihood of aspiration pneumonia. Successful gavage requires a high level of technical expertise (Murphy et al. 2001) to avoid inadvertent penetration of the trachea, oesophagus or stomach wall and inadvertent delivery of fluid into the thoracic or peritoneal cavity. It is widely stated (Smith & Kelleher 1973, Waynforth & Flecknell 1992, Brown et al. 2000, Watanabe et al. 2003, Balcombe et al. 2004) that repeated passage of a feeding tube or needle increases the probability of damage to the mouth, tongue, oesophagus and/or stomach, but we were unable to determine the incidence of such complications.

Little is known of the adverse physiological consequences of the use of gavage and other techniques of supplementation in newborn, juvenile or adult rats (Brown et al. 2000, Alban et al. 2001, Murphy et al. 2001, Bonnichsen et al. 2005). Gavage is known to induce transient increases of blood pressure, heart rate and body temperature in adult rats (Bonnichsen et al. 2005), and the gavage of large volumes of fluid (≥40 mL/kg) has greater impact than that of smaller volumes on heart rate (Alban et al. 2001) and plasma corticosterone concentrations (Brown et al. 2000).

2.1.4 Strategies for reducing the adverse effects of supplementation with bioactives

Maternal distress related to removal of the pups may be reduced by first transferring both dam and pups to a holding cage before removal and by subsequently returning the pups to the nest before the dam is returned (Hard 1975). Maternal rejection, infanticide or cannibalism may be reduced by ensuring that each pup is treated in a similar way and by covering the pups in the bedding and litter of the home cage prior to the return of the dam (Hard 1975, Poole 1987). Maternal sedation has also been used to prevent cannibalism of 2–5-day-old rats after surgery (Hayek & Kuehn 1982), but this may pose risks to the health of the dam if used on a regular basis.

Maternal habituation conducted prior to the birth of the pups is also said to reduce stress. ‘Hand gentling’ involves graded increases in daily petting and handling of the dam and is said to reduce the stress of oral delivery or gavage (Libbin & Person 1979, Libbin et al. 1982). Routine handling during cage cleaning and weighing may have a similar beneficial effect (Reynolds 1981, DeSantis & Schmaltz 1984).

Mortality from gavage may be reduced when it is performed under light anaesthesia but this may increase the likelihood of aspiration. Thus, while light halothane anaesthesia reduced the number of gavage-related deaths in adult rats, gavaged fluid often refluxed into the animal's mouth and/or nose during the procedure (Murphy et al. 2001). The use of such procedures on neonates is likely to be hazardous owing to their low body mass and the limited information regarding the effects of anaesthetic agents in this regard.

2.2 Substitution models

Substitution models involve artificial rearing in the absence of the dam. The young are reared either as a litter (Hoshiba 1986, 1996, Moriguichi et al. 2004) or individually (Messer et al. 1969, Hall 1975, Blake et al. 1988). The investigator must provide appropriate quantities of nutrients, appropriate environmental temperature and humidity, and appropriate substitutes for maternal activity to maintain cleanliness, stimulate urination and defaecation, and optimize sensory development.

Prolonged separation of pups from the mothr in substitution models is likely to be accompanied by profound physiological changes. Rat pups are suckled virtually continuously over the first few days, as the mother is absent for short periods only (Ader & Grota 1970). Unduly long periods of maternal absence bring about physiological changes which include: decreases in the rate of synthesis of ornithine decarboxylase (a key enzyme in polyamine biosynthesis) (Schipper & Verhofstad 2002) and of DNA, increases in plasma corticosterone concentrations, enhancement of the responsiveness of the hypothalamic–pituitary–adrenal axis to stressors and decreases in cellular responsiveness to growth hormone, insulin and prolactin (e.g. Kuhn et al. 1978, 1990, Schanberg & Kuhn 1985, Levine et al. 1991, Rosenfeld et al. 1991, 1992). Some of these changes are transient (Anderson & Schanberg 1975, Cirulli et al. 1992), provided that separation from the mother is not prolonged. However, an increasing body of evidence suggests that repeated periods of separation during the preweaning period produce long-lasting physiological changes that persist into adulthood (Kuhn & Schanberg 1998, Gonzalez et al. 2001, Levy et al. 2003, Lovic & Fleming 2004, Yamazaki et al. 2005, Lomanowska et al. 2006, Melo et al. 2006).

2.2.1 Modes of rearing

2.2.1.1 Rearing as a litter

Litters have been reared using conventional ‘shoe-box’ rat containers with supplementary heating (Gustafsson 1948, Pleasants 1959, Miller & Dymsza 1963, Berseth et al. 1983) and using automatic feeder housing systems (Hoshiba 1986, 1996, Moriguichi et al. 2004; see also text under the heading Delivery into the buccal cavity, below).

2.2.1.2 Rearing in isolation

Rearing of single pups generally requires more complex methodologies. Most reported studies are based on the ‘pup in a cup’ technique (Hall 1975). In general, each pup is housed in a lidded styrofoam beverage container floating in a temperature-controlled waterbath that maintains both heat and humidity. The containers are held in place by a grid mounted on top of the bath and are also anchored from below with weights. Each cup can move freely within the grid with the movement of the pup.

The rat pups are generally cannulated, the cannula exiting the container lid to be connected to a syringe driver that is housed in a refrigerator to prevent deterioration of the milk. Supplementation techniques are labour-intensive, as they require daily recharging of the syringes, cleaning of the feeding system, renewal of the bedding and anogenital stimulation of the pups to encourage voiding of urine and faeces.

2.2.2 Administration of milk substitutes

2.2.2.1 Delivery into the buccal cavity

Fluids may be delivered into the buccal cavity either by an automatic teat-feeder or by an implanted cannula. In the former, a silicone teat projects horizontally (Hoshiba 1986) or vertically downward (Hoshiba 1996, Moriguichi et al. 2004) into the rearing box from a nursing bottle. The area surrounding each teat is covered with a nylon-based fur material to mimic the body hair of the dam. The individual nursing bottles are recharged from a reservoir by a peristaltic pump.

A length of polyethylene tubing (diameter: ∼0.6 mm external; ∼0.28 mm internal) fitted with a disc-shaped flange is used for buccal cannulation. Typically, the pups are anaesthetized with an inhaled agent. The tip of the cannula is introduced into the buccal aspect of a puncture wound made by a needle (generally 22G) or steel wire and subsequently drawn through so that the flange seals and anchors it against the inner surface of the buccal cavity. The free end of the cannula is then ‘tunnelled’ subcutaneously to emerge from the skin at the nape of the pup's neck where it is secured in position with a washer or a drop of surgical cement. Such cannulae have been successfully inserted into the buccal cavity via the tongue (Hall & Rosenblatt 1977), the myelohyoid musculature in the floor of the mouth (Hall 1979), the hard palate from the nasal cavity (Blake et al. 1988) and a lateral cheek (Rudy & Hyson 1982).

2.2.2.2 Delivery into the oesophagus

An oesophageal cannula is advanced from the mouth to a point approximately halfway down the oesophagus as the conscious animal swallows. The proximal end of the cannula is then secured by either being glued to a lip (Moore et al. 1986) or looped around one corner of the mouth and passed across a cheek to the nape of the neck where it is secured with glue and covered with adhesive tape (Nuesslein & Schmidt 1990).

Pups may also be fed by frequent gavage (Gustafsson 1948, Pleasants 1959, Miller & Dymsza 1963, Berseth et al. 1983), typically three hourly for the first week, with the frequency reducing with increasing age.

2.2.2.3 Delivery into the stomach

A gastric cannula may be installed surgically under general anaesthetic by the direct (Messer et al. 1969) or the oral (Hall 1975) route. The direct route accesses the stomach via the anterior abdominal and stomach wall. Typically, the flanged end of the cannula is introduced into the fundal region of the stomach. Following closure of the incision, the projecting distal end of the cannula is advanced between the abdominal muscle layers to the dorsum of the lumbar spine where it is passed through the skin and secured with an external washer.

In the method using the oral route (Hall 1975), an orogastric tube is passed into the stomach lumen and a sharp-tipped lubricated stainless steel guide wire is advanced along it until the sharpened distal tip penetrates the stomach wall, peritoneum and abdominal wall and emerges at the left flank caudal to the most posterior rib. The sharpened tip of the guide wire is held in place while the orogastric tube is removed. The unflanged end of a flanged gastric cannula is then secured to the proximal end of the guide wire. The distal end of the guide wire is pulled through the fistula so that the attached cannula is pulled to the point where the proximally mounted flange is drawn down against the gastric mucosa. An external flange is threaded over the free end of the cannula and glued against the skin at the point of emergence. The free end of the cannula is then tunnelled subcutaneously to the nape of the neck so as to avoid direct traction on the fistula.

2.2.3 Milk substitution formulae

Rat milk contains an array of proteins, peptides, oligosaccharides, hormones, growth factors, immunoglobulins and other biologically active substances in appropriate proportions. These factors are known to facilitate digestion and absorption, immune protection and to promote growth (Severin & Wenshui 2005). Collection of natural rat milk is difficult and time-consuming (Brake 1979, Keen et al. 1980a,b, Tonkiss et al. 1987); moreover, serial milking significantly affects the composition of the milk (Keen et al. 1980a). Thus, various substitutes and formulations have been used including those based on the constituents of bovine milk (Gustafsson 1948, Pleasants 1959, Miller & Dymsza 1963, Messer et al. 1969, Smart et al. 1984, Auestad et al. 1989).

Bovine milk differs from rat milk in carbohydrate and protein content (Messer et al. 1969) and has an osmolarity twice that of rat milk (Miller & Czajka 1967). These differences are thought to account for disproportionate growth of the GI tract (Smart et al. 1983, 1984, Tonkiss et al. 1985, 1987, Kanno et al. 1997), to slowing of brain growth (Tonkiss et al. 1987, Auestad et al. 1989, Dvorak et al. 2000) and to abdominal distension (Tonkiss et al. 1987; see also text under the heading Abdominal distension, below) in artificially-reared rat pups. The composition of bovine milk may also adversely affect neonatal digestive processes. Thus, the high β-lactoglobulin content of bovine milk may alter its curding characteristics in the rodent GI tract. Similarly, differing proportions of antimicrobial components such as lactoferrin and IgG or IgA may influence the establishment of populations of gut microflora and alter microbial ecology (Yajima et al. 2001, Inoue & Ushida 2003, Nakayama et al. 2003). While these effects may be ameliorated by formulations with bovine milk components in similar proportions to those found in rat milk (Moore et al. 1986, Hiremagalur et al. 1992, Kanno et al. 1997), this will not obviate any effects of fundamental species-specific differences in the molecular structures of these components.

2.2.4 Strengths

The strength of substitution models depends on the extent to which they replicate the neonatal environment and diet in the absence of maternal influences. Substitution models may also be useful for evaluating the effects of delivery of nutrients to particular locations in the GI tract. Thus, comparison of the effects of delivery into the buccal cavity with delivery into the stomach may be useful for the study of the development of taste and dietary preference, ingestion and appetitive conditioning (Johanson & Hall 1979, Caza & Spear 1982, Johanson et al. 1984, Brake et al. 1986, Nizhnikov et al. 2002, Petrov et al. 2004, Myers et al. 2005). Substitution models have been used extensively in assessing the development of behaviour in the absence of the dam (e.g. Young & Dawson 1988, Hofer 1994, Caldji et al. 2000, Levy et al. 2003, Lovic & Fleming 2004). Substitution models may also be used to assess the role of bioactives in restoring normal gut growth and function after adverse events, and in identifying milk constituents having therapeutic value.

2.2.5 Problems

The use of substitution models has been complicated by high mortality rates, injury, infection, abnormal organ growth, abdominal distension and effects arising from isolation of individual pups from the dam and siblings.

2.2.5.1 Mortality rates

Although survival rates provide a view of animals' ability to survive the conditions of the model, it should be borne in mind that they do not assess the model's validity, particularly with respect to the maintenance of good health. Significant mortality does however cast doubt on a model's validity and the results arising from its use.

The overall mortality rates of such orally fed rat pups vary widely (0–12%) and precise causes of death are rarely established. Reported co-morbidities include abdominal distension (discussed below) and aspiration of milk into the lungs. Mortality rates of pups fed by buccal cannulae have not been reported, apart from enumeration of pups removed from the study because of various technical difficulties. Attendant technical difficulties include dislodgement, leakage or blockage of the cannula (Smith & Anderson 1984, Blake et al. 1988, Auestad et al. 1989, Philipps et al. 1997). Mortality rates of pups fed by gastric cannulae vary between 0% and 80%. Deaths are variously attributed to surgical trauma during cannula placement, internal bleeding, postoperative mortality, respiratory compromise secondary to abdominal distension and from technical difficulties related to cannula maintenance (Smith & Anderson 1984, Haney et al. 1986, Auestad et al. 1989, Hiremagalur et al. 1992).

2.2.5.2 Injury

The mouth and stomach are common sites of injury during cannulation and include perforation of the stomach wall, adjacent viscera or blood vessels. Poor technique and inexperience of the operator increase the likelihood of injury at installation (Morton et al. 2003). Subsequent growth, increasing physical activity of the pup, increasing tension on feeding lines and chewing by mother or pup may lead to cannula dislodgement and adjacent tissue damage from the cannula (Smith & Anderson 1984, Kanno et al. 1997, Myers et al. 2005).

The presence of an indwelling gastric cannula may alter GI motility (Soulsby et al. 2006) and subsequent gut development (Sangild et al. 2000) or lead to gastric inflammation and the development of ileus, acute dilation and perforation (Dickinson & Bisno 1989).

2.2.5.3 Infection

Although antenatal development of gastric parietal cell activity suggests that postnatal gastric acid secretion has the potential to provide some protection against GI colonization by pathogenic microorganisms (Johnson 1985), gastric acidity is not evident in rats at birth (Walthall et al. 2005). Moreover, the newborn is in a state of immune compromise and takes time to ingest and benefit from maternal antibodies (Renegar & Small 1999, Zinkernagel 2001). Accordingly, cannulation during the early neonatal period may provide a significant portal for secondary infection. Unrecognized neonatal infections may induce organ dysfunction (Gregory 1998) and compromise the biological validity of the model.

2.2.5.4 Abnormal growth and weight gain

A number of substitution models invoke body weight increases that parallel those of maternally-reared rat pups (Messer et al. 1969, Diaz et al. 1981, 1982, Smart et al. 1983, 1984, Tonkiss et al. 1985, 1987, Hoshiba 1986, Kojima et al. 1998) as objective and reliable indicators of health (Morton & Griffiths 1985, Hawkins 2002). However, they do not constitute proof of normal development or the ontogenic accumulation of normal tissues. Increases in body mass may result from conditions that lead to retention of body fluids such as hypoproteinaemia, cardiac oedema and abdominal ascites (Redgate et al. 1991, Bosch-Marce et al. 1999, Morton et al. 1999, Ullman-Cullere & Foltz 1999, Mangialardi et al. 2000, Gentilini et al. 2002, Bekheirnia & Schrier 2006). Increases in body mass may also result from selective hypertrophy of segments of the GI tract, liver, kidneys or spleen (Diaz et al. 1981, Smart et al. 1983, 1984, Tonkiss et al. 1985, 1987, Anderson & Smith 1987, Blake et al. 1988, Kanno et al. 1997, Kojima et al. 1998, Dvorak et al. 2000).

The differences in the growth rates of particular viscera have been variously attributed to differences in the mode of delivery of milk (Blake et al. 1988) and to differences between natural milk and the artificial formulations (Messer et al. 1969, Diaz et al. 1981, 1982, Auestad et al. 1989, Yajima et al. 1998, Dvorak et al. 2000). With regard to the latter hypothesis, differences in visceral growth rates have been variously attributed to the nutrient composition (Diaz et al. 1982, Smart et al. 1983, 1984, Tonkiss et al. 1985, Dvorak et al. 2000), physical characteristics (Diaz et al. 1981, 1982, Yeh & Holt 1986, Auestad et al. 1989, Kanno et al. 1997, Yajima et al. 1998, Kinouchi et al. 1999, Dvorak et al. 2000), gastric curd strength (Kanno et al. 1997, Yajima et al. 1998) and bacterial contamination (Auestad et al. 1989, Kanno et al. 1997) of artificial formulations. Other work has suggested that visceral growth rates may be selectively reduced by stress-induced increase in corticosteroid levels (Yeh et al. 1986), and by reduced levels of epidermal growth factor (Berseth 1987, Yajima et al. 1998), gastrin (Yeh et al. 1987), insulin (Yajima et al. 1998), insulin-like growth factor-I (Philipps et al. 1997) and triiodothyronine (Yeh et al. 1987).

2.2.5.5 Abdominal distension

Abdominal distension is a frequently reported problem in artificial rearing paradigms, generally resulting from an accumulation of gas, principally in the stomach and small intestine. The problem usually develops after 2–10 days of artificial rearing and has been variously attributed to high osmolality (Messer et al. 1969, Kanno et al. 1997) or inappropriate composition (Diaz et al. 1982, Tonkiss et al. 1987) of the milk formulation, to bacterial colonization of the GI tract (Kanno et al. 1997), and to accumulation of air from sucking on the artificial teat (Hoshiba 2004, Moriguichi et al. 2004). We have found no work describing an association between abdominal distension and postoperative infection after cannulation or to other changes in enteral microflora.

Untreated, abdominal distension may lead to respiratory compromise and death (Smart et al. 1984, Tonkiss et al. 1985). Recovery may be aided by reducing the infusion rate of the milk substitute, but this may limit growth (Smart et al. 1984, Tonkiss et al. 1985) so that the biological validity of results is questionable.

2.2.5.6 Separation from the mother

Although a number of physiological changes resulting from maternal absence may result from reduced intake of substances in maternal milk, some may result from reduction in maternal manipulations such as the licking of the pups. Stroking has been shown to regulate the secretion of growth hormone (Kuhn et al. 1978, 1990, Schanberg et al. 1984) and adrenocorticotrophic hormone (Suchecki et al. 1993, van Oers et al. 1998). Currently however, there are no published guidelines on the frequency and duration of such pup manipulations, particularly with regard to stimulation of urination and/or defaecation. It is possible that the frequency of manipulations used in substitution models to mimic such elements of maternal care may be too low, e.g. 12-hourly (e.g. Smart et al. 1983, Haney et al. 1986, Hofer et al. 1989, Hiremagalur et al. 1992, Kanno et al. 1997, Yajima et al. 1998, Dvorak et al. 2000).

2.2.5.7 Pain

Pain has been defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage (Merskey & Bogduk 1994). Severe and protracted pain will modify physiological and behavioural responses of animals (Craig et al. 1993, Mellor et al. 2000). The outgrowth of sensory axons from the dorsal root ganglia to the skin occurs antenatally in the rat (Reynolds et al. 1991, Karanth 1994, Jackman & Fitzgerald 2000), as in most vertebrates, so that the neonate is able to respond to noxious stimuli, although the responses may not be predictable or organized (Fitzgerald 1994). Recent evidence suggests that newborn and juvenile animals have signalling pathways, not found in the mature nervous system, that may enhance the experience of pain and that this circuitry may be modified by sensory inputs occurring during the early postnatal period (Fitzgerald 2005). Conversely, the postnatal ontogeny of the electroencephalogram suggests that the development of the central nervous system may not reach a sufficient level of organization to enable full pain perception until about five days after birth or more (Ellingson & Rose 1970).

While it seems likely that surgical cannulation procedures and distress from maternal separation may bring about pain, there are currently no means of validating this or of relating it to deleterious effects in relation to general indices of wellbeing such as weight gain.

2.2.6 Procedural refinements

A number of refinements to substitution models have reduced mortality and possibly morbidity (not reported). Thus, orogastric (Hall 1975) rather than transabdominal gastric cannulation (Messer et al. 1969) reportedly gives faster recovery and lower overall mortality rates (Hall 1975).

Nevertheless, it is important to consider the size of the animal in relation to the cannula size, given the general relationship between wound size and postoperative mortality and morbidity (Jaffray 2005). Thus, in a 60 mm long rat pup, although insertion of a cannula with an external diameter of 0.6 mm will itself produce a wound that is only around 1% of the animal's total length, this represents a proportionately much greater wound size at the site of cannulation (e.g. in the cheek, hard palate or stomach). The procedure used to position and secure such a cannula (e.g. skin incision and tunnelling under the skin) is also likely to have significant postoperative effects.

The use of aseptic technique is known to reduce death rate, and improve postoperative health and the reliability of experimental outcomes (Popp & Brennan 1981, Park et al. 1992, Cunliffe-Beamer 1993). The use of effective anaesthesia and analgesia may similarly reduce postoperative mortality and morbidity (Flecknell 1999), although until recently few guidelines for anaesthesia of newborn or young rats have been available (Danneman & Mandrell 1997) and there are apparently none for postoperative analgesia.

3. Discussion

Judged on a basis of mortality, substitution models are generally less successful than maintenance models. Thus, the overall survival rate of rat pups in maintenance models ranges between 88% and 100%, while that of substitution models ranges between 20% and 100%. This is to be expected given that substitution models require satisfactory replacement of the behavioural, physiological and nutritional elements of the system in a situation that is devoid of maternal care, exacerbated by the current lack of detailed knowledge regarding the timing and relative contribution of these elements. Accordingly, results obtained from substitution models where there are suboptimal levels of survival should be viewed with caution, because in such experiments results of a manipulation may be confounded by suboptimal duplication of maternal care. Thus, in order to survive or to thrive, the animal may be required both to cope with the experimental manipulation and to exhibit a robust pathophysiological response to the deficiency in maternal care. In effect the experimental scenario may test pathophysiological robustness rather than responses reflecting normal development. Further, when a particular manipulation fails to achieve normal development, the experimenter may resort to the circular argument that this failure results from deficiencies inherent in the model.

Given the current incomplete state of knowledge regarding the optimal neonatal environment, it is necessary to ask whether environmental deficiencies are likely to generate significant stress to the animal. In the absence of any direct evaluation, it seems reasonable to assume significant ongoing stress in situations where mortality rates are high. In such situations experimental findings should be validated by active exclusion of pathological states by appropriate postmortem, histological and microbiological procedures, i.e. to provide proof of normalcy.

It is also important to identify any symptoms that may similarly signal departure from normalcy, e.g. pain. The use of scoring systems and measurement tools based on both physiological and/or behavioural responses to nociceptive inputs have been developed for use in the adults of several mammalian species (Morton & Griffiths 1985, Morton et al. 1999, Mellor et al. 2000, Flecknell & Roughan 2004). Given the differences in the neurological development and responses of neonates of different species, it may be necessary to refine these methods for use in immature rats.

Cannulation is necessary in substitution methodology to avoid frequent forceful manipulations. However, the pain that may be generated by cannulation procedures, although often short-lived, may also adversely influence subsequent development. We found no published studies comparing the development of newborn or young rats subsequent to cannulation with and without analgesia/anaesthesia.

Maintenance models are not as prone to the procedural doubts and the potential welfare problems that surround the use of substitution models, but do have specific methodological limitations. Thus, in current models the operator is limited to the premature or supramaximal dosage of maternally-suckled pups with active agents. However, given the current upswing in the availability of genetic ‘knockout’ animals and the possibility of cross fostering of normal pups onto ‘knockout’ mothers it is likely that such limitations will be removed in the near future. Knockout technology has provided tremendous insight into developmental processes and the availability of a maternal gene knockout model would benefit the investigation of bioactive compounds on GI physiology and the welfare of the animals involved. The use of supplementation models in the current form is hampered by a lack of knowledge regarding the cumulative effects of maternal deprivation on neonatal physiology. The use of indwelling cannulae to avoid such effects may be justified only provided that they have lesser adverse effect on neonatal physiology and maternal care than does maternal deprivation.

In conclusion, we recommend that the following strategies be applied to the use of newborn and juvenile rats as models for researching GI development. The models should use or duplicate the ‘natural state’ of rearing as far as practicable. Detailed comparisons should be made between ‘model’ pups and ‘maternally-reared’ pups, and additional control groups used as appropriate. Invasive interventions should be avoided where possible and the deleterious consequences of necessary interventions minimized. The biological validity of the model should be confirmed through the identification and use of indices of normalcy and good welfare status in the pups in order to ensure that scientifically meaningful results are obtained. Finally, it is expected that in the relatively near future the availability of genetic ‘knockout’ female rats will facilitate application of these strategies.

Footnotes

Acknowledgements

The authors are grateful to all of those who provided advice based on their direct experience of different aspects of the models evaluated in this review, in particular Ms Anne Broomfield (Massey University, New Zealand), Dr Ruth Napper (University of Otago, New Zealand) and Dr Junji Hoshiba (Okayama University, Japan).

References

Ader

, Grota

(1970) Rhythmicity in maternal behaviour of Rattus norvegicus. Animal Behaviour 18, 144–50

Alban

, Dahl

, Hansen

, (2001) The welfare impact of increased gavaging doses in rats. Animal Welfare 10, 303–14

Alberts

(1978) Huddling by rat pups: multisensory control of contact behavior. Journal of Comparative and Physiological Psychology 92, 220–30

Anderson

, Schanberg

(1975) Effect of thyroxine and cortisol on brain ornithine decarboxylase activity and swimming behavior in developing rat. Biochemical Pharmacology 24, 495–501

Anderson

, Smith

(1987) The effects of maternal isolation and light and feeding cycles on the growth of rat pups. Developmental Psychobiology 20, 245–59

Auestad

, Korsak

, Bergstrom

, Edmond

(1989) Milk-substitutes comparable to rat's milk: their preparation, composition and impact on development and metabolism in the artificially reared rat. British Journal of Nutrition 61, 495–518

Balcombe

, Barnard

, Sandusky

(2004) Laboratory routines cause animal stress. Contemporary Topics in Laboratory Animal Science 43, 42–51

Bekheirnia

, Schrier

(2006) Pathophysiology of water and sodium retention: edematous states with normal kidney function. Current Opinion in Pharmacology 6, 202–7

Berseth

(1987) Enhancement of intestinal growth in neonatal rats by epidermal growth factor in milk. American Journal of Physiology 253, G662–5

10.

Berseth

, Lichtenberger

, Morriss

(1983) Comparison of the gastrointestinal growth promoting effects of rat colostrum and mature milk in newborn rats in vivo . American Journal of Clinical Nutrition 37, 52–60

11.

Blake

, Lau

, Henning

(1988) A new method for the artificial raising of infant rats: the palate cannula. Physiology and Behavior 42, 495–8

12.

Bockamp

, Maringer

, Spangenberg

, (2002) Of mice and models: improved animal models for biomedical research. Physiological Genomics 11, 115–32

13.

Bonnichsen

, Dragsted

, Hansen

(2005) The welfare impact of gavaging laboratory rats. Animal Welfare 14, 223–7

14.

Bosch-Marce

, Poo

, Jimenez

, (1999) Comparison of two aquaretic drugs (Niravoline and OPC-31260) in cirrhotic rats with ascites and water retention. Journal of Pharmacology and Experimental Therapeutics 289, 194–201

15.

Brake

(1979) Procedures for the collection of milk from the rat dam. Physiology and Behavior 22, 795–7

16.

Brake

, Tavana

, Myers

(1986) A method for recording and analyzing intraoral negative pressure in suckling rat pups. Physiology and Behavior 36, 575–8

17.

Brown

, Dinger

, Levine

(2000) Stress produced by gavage administration in the rat. Contemporary Topics in Laboratory Animal Science 39, 17–21

18.

Caldji

, Diorio

, Meaney

(2000) Variations in maternal care in infancy regulate the development of stress reactivity. Biological Psychiatry 48, 1164–74

19.

Caza

, Spear

(1982) Pharmacological manipulation of milk-induced behaviors in 3-day-old rat pups. Pharmacology Biochemistry and Behavior 16, 481–6

20.

Cirulli

, Gottlieb

, Rosenfeld

, Levine

(1992) Maternal factors regulate stress responsiveness in the neonatal rat. Psychobiology 20, 143–52

21.

Craig

, Whitfield

, Grunau

RVE

, (1993) Pain in the preterm neonate: behavioral and physiological indexes. Pain 52, 287–99

22.

Cunliffe-Beamer

(1993) Applying principles of aseptic surgery to rodents. Animal Welfare Information Center Newsletter 4, 3–6

23.

Danneman

, Mandrell

(1997) Evaluation of five agents/methods for anesthesia of neonatal rats. Laboratory Animal Science 47, 386–95

24.

Del Prado

, Delgado

, Villalpando

(1997) Maternal lipid intake during pregnancy and lactation alters milk composition and production and litter growth in rats. Journal of Nutrition 127, 458–62

25.

DeSantis

, Schmaltz

(1984) The mother–litter relationship in developmental rat studies: cannibalism vs. caring. Developmental Psychobiology 17, 255–62

26.

Diaz

, Moore

, Petracca

, (1981) Artificial rearing of preweanling rats: the effectiveness of direct intra-gastric feeding. Physiology and Behavior 27, 1103–5

27.

Diaz

, Moore

, Petracca

, (1982) Artificial rearing of rat pups with a protein-enriched formula. Journal of Nutrition 112, 841–7

28.

Dickinson

, Bisno

(1989) Infections associated with indwelling devices: concepts of pathogenesis; infections associated with intravascular devices. Antimicrobial Agents and Chemotherapy 33, 597–601

29.

Ellingson

, Rose

(1970) Ontogenesis of the electroencephalogram. In: Developmental Neurobiology ( Himwich

, ed). Springfield: Charles C Thomas, 441–74

30.

Dvorak

, McWilliam

, Williams

, (2000) Artificial formula induces precocious maturation of the small intestine of artificially reared suckling rats. Journal of Pediatric Gastroenterology and Nutrition 31, 162–9

31.

Fitzgerald

(2005) The development of nociceptive circuits. Nature Reviews Neuroscience 6, 507–20

32.

Fitzgerald

(1994) Neurobiology of fetal and neonatal pain. In: Textbook of Pain. 3rd edn. ( Wall

, Melzack

, eds). Edinburgh: Churchill Livingstone, 153–63

33.

Flecknell

(1999) Pain: assessment, alleviation and avoidance in laboratory animals. ANZCCART News 12, 1–10

34.

Flecknell

, Roughan

(2004) Assessing pain in animals – putting research into practice. Animal Welfare 13, S71–5

35.

Forbes

, Tracy

, Resnick

, Morgane

(1977) Effects of maternal dietary protein restriction on growth of brain and body in rat. Brain Research Bulletin 2, 131–5

36.

Gentilini

, Vizzutti

, Gentilini

, (2002) Update on ascites and hepatorenal syndrome. Digestive and Liver Disease 34, 592–605

37.

Gonzalez

, Lovic

, Ward

, (2001) Intergenerational effects of complete maternal deprivation and replacement stimulation on maternal behavior and emotionality in female rats. Developmental Psychobiology 38, 11–32

38.

Gregory

(1998) Physiological mechanisms causing sickness behaviour and suffering in diseased animals. Animal Welfare 7, 293–305

39.

Gustafsson

(1948) Germ-free rearing of rats. Acta Pathologica et Microbiologica Scandinavica Supplementum 73, 1–130

40.

Hall

(1975) Weaning and growth of artificially reared rats. Science 190, 1313–15

41.

Hall

(1979) Feeding and behavioral activation in infant rats. Science 205, 206–9

42.

Hall

(1990) The ontogeny of ingestive behaviour. In: Neurobiology of Food and Fluid Intake ( Stricker

, ed). New York: Plenum Press, 77–123

43.

Hall

, Rosenblatt

(1977) Suckling behavior and intake control in developing rat pup. Journal of Comparative and Physiological Psychology 91, 1232–47

44.

Haney

, Estrin

, Caliendo

, Patel

(1986) Precocious induction of hepatic glucokinase and malic enzyme in artificially reared rat pups fed a high carbohydrate diet. Archives of Biochemistry and Biophysics 244, 787–94

45.

Hard

(1975) Thymectomy in the neonatal rat. Laboratory Animals 9, 105–10

46.

Hawkins

(2002) Recognizing and assessing pain, suffering and distress in laboratory animals: a survey of current practice in the UK with recommendations. Laboratory Animals 36, 378–95

47.

Hayek

, Kuehn

(1982) A method for preventing maternal cannibalism after neonatal rat surgery. Laboratory Animal Science 32, 171–2

48.

Henning

(1986) Development of the gastrointestinal tract. Proceedings of the Nutrition Society 45, 39–44

49.

Hiremagalur

, Johanning

, Kalhan

, Patel

(1992) Alterations in hepatic lipogenic capacity in rat pups artificially reared on a milk-substitute formula high in carbohydrate or medium-chain triacylglycerides. Journal of Nutritional Biochemistry 3, 474–80

50.

Hofer

(1994) Early relationships as regulators of infant physiology and behavior. Acta Paediatrica 83, 9–18

51.

Hofer

, Shair

, Murowchick

(1989) Isolation distress and maternal comfort responses of 2-week-old rat pups reared in social isolation. Developmental Psychobiology 22, 553–66

52.

Hoshiba

(1986) An automatic feeder for infant rats. Laboratory Animal Science 36, 682–5

53.

Hoshiba

(1996) Automatic feeder for newborn rat use within 12 hours of birth. Contemporary Topics in Laboratory Animal Science 35, 83–6

54.

Hoshiba

(2004) Method for hand-feeding mouse pups with nursing bottles. Contemporary Topics in Laboratory Animal Science 43, 50–3

55.

Inoue

, Ushida

(2003) Development of the intestinal microbiota in rats and its possible interactions with the evolution of the luminal IgA in the intestine. FEMS Microbiology Ecology 45, 147–53

56.

Jackman

, Fitzgerald

(2000) Development of peripheral hindlimb and central spinal cord innervation by subpopulations of dorsal root ganglion cells in the embryonic rat. Journal of Comparative Neurology 418, 281–98

57.

Jaffray

(2005) Minimally invasive surgery. Archives of Disease in Childhood 90, 537–42

58.

Johanson

, Hall

(1979) Appetitive learning in 1-day-old rat pups. Science 205, 419–21

59.

Johanson

, Hall

, Polefrone

(1984) Appetitive conditioning in neonatal rats – conditioned ingestive responding to stimuli paired with oral infusions of milk. Developmental Psychobiology 17, 357–81

60.

Johnson

(1985) Functional development of the stomach. Annual Review of Physiology 47, 199–215

61.

Kanno

, Koyanagi

, Katoku

, (1997) Simplified preparation of a refined milk formula comparable to rat's milk: influence of the formula on development of the gut and brain in artificially reared rat pups. Journal of Pediatric Gastroenterology and Nutrition 24, 242–52

62.

Karanth

(1994) Ontogeny of nerves and neuropeptides in the skin of rat: an immunocytochemical study. Experimental Dermatology 3, 171–5

63.

Keen

, Lonnerdal

, Sloan

, Hurley

(1980a) Effect of dietary iron, copper and zinc chelates of nitrilotriacetic acid (Nta) on trace metal concentrations in rat milk and maternal and pup tissues. Journal of Nutrition 110, 897–906

64.

Keen

, Lonnerdal

, Sloan

, Hurley

(1980b) Effects of milking procedure on rat milk composition. Physiology and Behavior 24, 613–15

65.

Kehoe

, Shoemaker

, Triano

, (1996) Repeated isolation in the neonatal rat produces alterations in behavior and ventral striatal dopamine release in the juvenile after amphetamine challenge. Behavioral Neuroscience 110, 1435–44

66.

Kinouchi

, Koizumi

, Kuwata

, Yajima

(1999) Evaluation of the development of intestinal function in rats reared on hydrolyzed or native protein-based milk formula. Journal of Pediatric Gastroenterology and Nutrition 29, 155–62

67.

Kliewer

, Rasmussen

(1987) Malnutrition during the reproductive cycle: effects on galactopoietic hormones and lactational performance in the rat. American Journal of Clinical Nutrition 46, 926–35

68.

Kojima

, Nishimura

, Yajima

, (1998) Effect of intermittent feeding on the development of disaccharidase activities in artificially reared rat pups. Comparative Biochemistry and Physiology A: Molecular and Integrative Physiology 121, 289–97

69.

Koldovsky

(1989) Search for role of milk-borne biologically-active peptides for the suckling. Journal of Nutrition 119, 1543–51

70.

Korotkova

, Gabrielsson

, Hanson

, Strandvik

(2001) Maternal essential fatty acid deficiency depresses serum leptin levels in suckling rat pups. Journal of Lipid Research 42, 359–65

71.

Kuhn

, Butler

, Schanberg

(1978) Selective depression of serum growth hormone during maternal deprivation in rat pups. Science 201, 1034–6

72.

Kuhn

, Pauk

, Schanberg

(1990) Endocrine responses to mother-infant separation in developing rats. Developmental Psychobiology 23, 395–410

73.

Kuhn

, Schanberg

(1998) Responses to maternal separation: mechanisms and mediators. International Journal of Developmental Neuroscience 16, 261–70

74.

Lau

, Simpson

(2004) Animal models for the study of the effect of prolonged stress on lactation in rats. Physiology and Behavior 82, 193–7

75.

Levine

, Huchton

, Wiener

, Rosenfeld

(1991) Time course of the effect of maternal deprivation on the hypothalamic-pituitary-adrenal axis in the infant rat. Developmental Psychobiology 24, 547–58

76.

Levy

, Melo

, Galef

, (2003) Complete maternal deprivation affects social but not spatial learning in adult rats. Developmental Psychobiology 43, 177–91

77.

Libbin

, Person

(1979) Neonatal rat surgery: avoiding maternal cannibalism. Science 206, 66

78.

Libbin

, Person

, Ayala

(1982) Procedures for the use of neonatal rats in surgical research. Journal of Surgical Research 33, 519–23

79.

Lim

, Hoshiba

, Moriguchi

, Salem

(2005) N-3 fatty acid deficiency induced by a modified artificial rearing method leads to poorer performance in spatial learning tasks. Pediatric Research 58, 741–8

80.

Lomanowska

, Rana

, McCutcheon

, (2006) Artificial rearing alters the response of rats to natural and drug-mediated rewards. Developmental Psychobiology, 48, 301–14

81.

Lovic

, Fleming

(2004) Artificially-reared female rats show reduced prepulse inhibition and deficits in the attentional set shifting task: reversal of effects with maternal-like licking stimulation. Behavioural Brain Research 148, 209–19

82.

Mangialardi

, Martin

, Bernard

, (2000) Hypoproteinemia predicts acute respiratory distress syndrome development, weight gain, and death in patients with sepsis. Critical Care Medicine 28, 3137–45

83.

Matias

, Leonhardt

, Lesage

, (2003) Effect of maternal under nutrition on pup body weight and hypothalamic endocannabinoid levels. Cellular and Molecular Life Sciences 60, 382–9

84.

Mellor

, Cook

, Stafford

(2000) Quantifying some responses to pain as a stressor. In: The Biology of Animal Stress: Basic Principles and Implications for Welfare ( Moberg

, Mench

, eds) Oxon: CAB International, 171–98

85.

Melo

, Lovic

, Gonzalez

, (2006) Maternal and littermate deprivation disrupts maternal behavior and social-learning of food preference in adulthood: tactile stimulation, nest odor, and social rearing prevent these effects. Developmental Psychobiology 48, 209–19

86.

Melton

(1994) Gene targeting in the mouse. Bioessays 16, 633–8

87.

Merskey

, Bogduk

(1994) Classification of chronic pain. In: IASP Task Force on Taxonomy. 2nd edn. Seattle: IASP Press, 209–14

88.

Messer

, Thoman

, Therrasa

, Dallman

(1969) Artificial feeding of infant rats by continuous gastric infusion. Journal of Nutrition 98, 404–10

89.

Miller

, Czajka

(1967) Influence of dietary osmolarity on survival in neonatal rat. Biologia Neonatorum 11, 197–203

90.

Miller

, Dymsza

(1963) Artificial feeding of neonatal rats. Science 141, 517–18

91.

Montgomery

, Mulberg

, Grand

(1999) Development of the human gastrointestinal tract: twenty years of progress. Gastroenterology 116, 702–31

92.

Moore

, Greene

, Said

, (1986) Effect of epidermal growth factor and artificial feeding in suckling rats. Pediatric Research 20, 1248–51

93.

Morton

, Griffiths

PHM

(1985) Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Veterinary Record 116, 431–6

94.

Morton

, Ambrose

, Leach

, (1999) Adverse effects: recognition and assessment, and humane endpoints. In: Progress in the Reduction, Refinement and Replacement of Animal Experimentation: Proceedings of the Third World Congress on Alternatives and Animal Use in the Life Sciences ( Balls

, van Zeller

A-M

, Halder

, eds). Amsterdam: Elsevier Science, 1083–93

95.

Morton

, Hawkins

, Bevan

, (2003) Refinements in telemetry procedures: seventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement, Part A. Laboratory Animals 37, 261–99

96.

Muller

, Cox

(1946). Effect of changes in diet on volume and composition of rat milk. Journal of Nutrition 31, 249–59

97.

Murphy

, Smith

, Shaivitz

, (2001) The effect of brief halothane anesthesia during daily gavage on complications and body weight in rats. Contemporary Topics in Laboratory Animal Science 40, 9–12

98.

Myers

, Ferris

, Sclafani

(2005) Flavor preferences conditioned by postingestive effects of nutrients in preweanling rats. Physiology and Behavior 84, 407–19

99.

Nakayama

, Yajima

, Hatano

, (2003) Intestinal adherent bacteria and bacterial translocation in breast-fed and formula-fed rats in relation to susceptibility to infection. Pediatric Research 54, 364–71

100.

Nizhnikov

, Petrov

, Varlinskaya

, Spear

(2002) Newborn rats' first suckling experience: taste differentiation and suckling plasticity. Physiology and Behavior 76, 181–98

101.

Nuesslein

, Schmidt

(1990) Development of circadian cycle of core temperature in juvenile rats. American Journal of Physiology 259, R270–6

102.

Park

, Clegg

, Harveyclark

, Hollenberg

(1992) Improved techniques for successful neonatal rat surgery. Laboratory Animal Science 42, 508–13

103.

Petrov

, Nizhnikov

, Kozlov

, (2004) Repetitive exposures to a surrogate nipple providing nutritive and non-nutritive fluids: effects on suckling behavior of the newborn rat. Appetite 43, 185–94

104.

Philipps

, Anderson

, Dvorak

, (1997) Growth of artificially fed infant rats: effect of supplementation with insulin-like growth factor I. American Journal of Physiology 41, R1532–9

105.

Pleasants

(1959) Rearing germfree Cesarean-born rats, mice, and rabbits through weaning. Annals of the New York Academy of Sciences 78, 116–26

106.

Polan

, Hofer

(1999) Maternally directed orienting behaviors of newborn rats. Developmental Psychobiology 34, 269–79

107.

Poole

(1987) The UFAW Handbook on the Care and Management of Laboratory Animals. 6th edn. New York: Churchill Livingstone

108.

Popp

, Brennan

(1981) Long term vascular access in the rat: importance of asepsis. American Journal of Physiology 241, H606–12

109.

Rasmussen

(1998) Effects of under- and overnutrition on lactation in laboratory rats. Journal of Nutrition 128, 390–3

110.

Rasmussen

, Warman

(1983) Effect of maternal malnutrition during the reproductive cycle on growth and nutritional status of suckling rat pups. American Journal of Clinical Nutrition 38, 77–83

111.

Redgate

, Deutsch

, Boggs

(1991) Time of death of CNS tumor-bearing rats can be reliably predicted by body weight-loss patterns. Laboratory Animal Science 41, 269–73

112.

Renegar

, Small

(1999) Passive immunisation: systemic and mucosal. In: Mucosal Immunology ( Ogra

, Mestecky

, Lamm

, Strober

, Bienenstock

, McGhee

, eds). London: Academic Press, 729–38

113.

Reynolds

(1981) Preventing maternal cannibalism in rats. Science 213, 1146

114.

Reynolds

, Fitzgerald

, Benowitz

(1991) Gap-43 expression in developing cutaneous and muscle nerves in the rat hindlimb. Neuroscience 41, 201–11

115.

Rosenblatt

, Lehrman

(1963) Maternal behaviour of the laboratory rat. In: Maternal Behavior in Mammals ( Rheingold

, ed). New York: Wiley, 8–57

116.

Rosenfeld

, Gutierrez

, Martin

, (1991) Maternal regulation of the adrenocortical-response in preweanling rats. Physiology and Behavior 50, 661–71

117.

Rosenfeld

, Suchecki

, Levine

(1992) Multifactorial regulation of the hypothalamic-pituitary-adrenal axis during development. Neuroscience and Biobehavioral Reviews 16, 553–68

118.

Rudy

, Hyson

(1982) Consummatory response conditioning to an auditory stimulus in neonatal rats. Behavioral and Neural Biology 34, 209–14

119.

Sangild

, Fowden

, Trahair

(2000) How does the foetal gastrointestinal tract develop in preparation for enteral nutrition after birth? Livestock Production Sciences 66, 141–50

120.

Schanberg

, Evoniuk

, Kuhn

(1984) Tactile and nutritional aspects of maternal care: specific regulators of neuroendocrine function and cellular development. Proceedings of the Society for Experimental Biology and Medicine 175, 135–46

121.

Schanberg

, Kuhn

(1985) The biochemical effects of tactile deprivation in neonatal rats. Perspectives in Behavioral Medicine 2, 133–48

122.

Schank

, Alberts

(2000) The developmental emergence of coupled activity as cooperative aggregation in rat pups. Proceedings of the Royal Society of London Series B-Biological Sciences 267, 2307–15

123.

Schipper

, Verhofstad

AAJ

(2002) Distribution patterns of ornithine decarboxylase in cells and tissues: facts, problems, and postulates. Journal of Histochemistry and Cytochemistry 50, 1143–60

124.

Severin

, Wenshui

(2005) Milk biologically active components as nutraceuticals: review. Critical Reviews in Food Science and Nutrition 45, 645–56

125.

Sheard

, Walker

(1988) The role of breast-milk in the development of the gastrointestinal tract. Nutrition Reviews 46, 1–8

126.

Smart

, Stephens

, Katz

(1983) Growth and development of rats artificially reared on a high or a low plane of nutrition. British Journal of Nutrition 49, 497–506

127.

Smart

, Stephens

, Tonkiss

, (1984) Growth and development of rats artificially reared on different milk-substitutes. British Journal of Nutrition 52, 227–37

128.

Smith

(2006) Ontogeny of ingestive behavior. Developmental Psychobiology 48, 345–59

129.

Smith

, Kelleher

(1973) Method for intragastric feeding of neonatal rats. Laboratory Animal Science 23, 682–4

130.

Smith

, Anderson

(1984) Effects of maternal isolation on the development of activity rhythms in infant rats. Physiology and Behavior 33, 751–6

131.

Soulsby

, Khela

, Yazaki

, Evans

, Hennessy

, Powell-Tuck

(2006) Measurements of gastric emptying during continuous nasogastric infusion of liquid feed: electrical impedance tomography versus gamma scintigraphy. Clinical Nutrition 25, 671–80

132.

Stanton

, Gutierrez

, Levine

(1988) Maternal deprivation potentiates pituitary-adrenal stress responses in infant rats. Behavioral Neuroscience 102, 692–700

133.

Suchecki

, Rosenfeld

, Levine

(1993) Maternal regulation of the hypothalamic-pituitary-adrenal axis in the infant rat: the roles of feeding and stroking. Developmental Brain Research 75, 185–92

134.

Tonkiss

, Smart

, Auestad

, Edmond

(1985) Type of milk substitute influences growth of the gastrointestinal tract in artificially reared rat pups. Journal of Pediatric Gastroenterology and Nutrition 4, 818–25

135.

Tonkiss

, Smart

, Massey

(1987) Growth and development of rats artificially reared on rats milk or rats milk milk-substitute combinations. British Journal of Nutrition 57, 3–11

136.

Ullman-Cullere

, Foltz

(1999) Body condition scoring: a rapid and accurate method for assessing health status in mice. Laboratory Animal Science 49, 319–23

137.

Uvnas-Moberg

(1997) Physiological and endocrine effects of social contact. In: Integrative Neurobiology of Affiliation ( Carter

, Lederhendler

, Kirkpatrick

, eds). New York: New York Academy of Sciences, 146–63

138.

van Oers

HJJ

, de Kloet

, Whelan

, Levine

(1998) Maternal deprivation effect on the infant's neural stress markers is reversed by tactile stimulation and feeding but not by suppressing corticosterone. Journal of Neuroscience 18, 10171–9

139.

Walthall

, Cappon

, Hurtt

, Zoetis

(2005) Postnatal development of the gastrointestinal system: a species comparison. Birth Defects Research. Part B, Developmental and Reproductive Toxicology 74, 132–56

140.

Watanabe

, Kuwagata

, Yoshimura

, (2003) An improved technique for repeated gavage administration to rat neonates. Congenital Anomalies 43, 177–9

141.

Waynforth

, Flecknell

(1992) Intragastric administration by gavage. In: Experimental and Surgical Technique in the Rat. 2nd edn. ( Waynforth

, Flecknell

, eds). London: Academic Press, 27–32

142.

Weaver

, Desai

, Austin

, (1998) Effects of protein restriction in early life on growth and function of the gastrointestinal tract of the rat. Journal of Pediatric Gastroenterology and Nutrition 27, 553–9

143.

(1996) Development of the newborn GI tract and its relation to colostrum milk intake: a review. Reproduction Fertility and Development 8, 35–48

144.

Yajima

, Kanno

, Katoku

, Kuwata

(1998) Gut hypertrophy in response to the ratios of casein and whey protein in milk formulas in artificially reared rat pups. Biology of the Neonate 74, 314–22

145.

Yajima

, Nakayama

, Hatano

, (2001) Bacterial translocation in neonatal rats: the relation between intestinal flora, translocated bacteria, and influence of milk. Journal of Pediatric Gastroenterology and Nutrition 33, 592–601

146.

Yamazaki

, Ohtsuki

, Yoshihara

, (2005) Maternal deprivation in neonatal rats of different conditions affects growth rate, circadian clock, and stress responsiveness differentially. Physiology and Behavior 86, 136–44

147.

Yeh

, Du

, Holt

(1986) Endogenous corticosterone rather than dietary sucrose as a modulator for intestinal sucrase activity in artificially reared rat pups. Journal of Nutrition 116, 1334–42

148.

Yeh

, Holt

(1986) Ontogenic timing mechanism initiates the expression of rat intestinal sucrase activity. Gastroenterology 90, 520–6

149.

Yeh

, Yeh

, Holt

(1987) Hormonal regulation of adaptive intestinal growth in artificially reared rat pups. American Journal of Physiology 253, G802–8

150.

Young

, Dawson

(1988) Effects of environmental temperature on the development of a noradrenergic thermoregulatory mechanism in the rat. Pflugers Archiv: European Journal of Physiology 412, 141–6

151.

Zan

, Haag

, Chen

, (2003) Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nature Biotechnology 21, 645–51

152.

Zinkernagel

(2001) Maternal antiobodies, childhood infections and autoimmune diseases. New England Journal of Medicine 345, 1331–5

An appraisal of the strengths and weaknesses of newborn and juvenile rat models for researching gastrointestinal development

Abstract

Keywords

1. Introduction

2. Materials and methods

2.1 Maintenance models

2.1.1 Supplementation techniques

2.1.1.1 Oral delivery

2.1.1.2 Oesophageal gavage

2.1.2 Strengths

2.1.3 Problems

2.1.4 Strategies for reducing the adverse effects of supplementation with bioactives

2.2 Substitution models

2.2.1 Modes of rearing

2.2.1.1 Rearing as a litter

2.2.1.2 Rearing in isolation

2.2.2 Administration of milk substitutes

2.2.2.1 Delivery into the buccal cavity

2.2.2.2 Delivery into the oesophagus

2.2.2.3 Delivery into the stomach

2.2.3 Milk substitution formulae

2.2.4 Strengths

2.2.5 Problems

2.2.5.1 Mortality rates

2.2.5.2 Injury

2.2.5.3 Infection

2.2.5.4 Abnormal growth and weight gain

2.2.5.5 Abdominal distension

2.2.5.6 Separation from the mother

2.2.5.7 Pain

2.2.6 Procedural refinements

3. Discussion

Footnotes

Acknowledgements

References