Pediatric Nutrit ion

Deborah S. Greco, DVM, PhD

KEYWORDS

� Bitch � Puppy � Queen � Kitten � Milk replacer � Probiotics � Prebiotics

KEY POINTS

� Feeding a balanced commercial diet to lactating and growing puppies and kittens pro-vides the necessary macronutrients (protein, fat, and so forth), vitamins, minerals, andsupplements required for normal growth and development.

� The most important immune-related function of good intestinal microflora is protectionagainst infection and colonization by harmful, and sometimes pathogenic, bacteria.

� Added benefit may arise from the use of docosahexaenoic acid–supplemented diets andfrom the use of prebiotic fibers, colostrum, and probiotics to promote growth and devel-opment of a healthy gastrointestinal tract, microbiota, and immune system in puppies andkittens.

INTRODUCTION

Balanced commercial dog foods designed for all life stages are the mainstay offeeding for optimal reproductive capacity in the bitch and ideal growth rates in puppiesand kittens. Recent evidence suggests that certain micronutrients and macronutri-ents, when balanced with other nutrients in the formulation, may provide a healthierimmune system, balanced gastrointestinal (GI) microbiota, and more acute hearingand vision.

PREGNANT AND LACTATING BITCHES

In general, pregnant and lactating bitches should be fed a high-energy (30% protein;20% fat on a dry matter basis [DMB]), highly digestible commercial dog food that isbalanced for vitamins and minerals. The food should be labeled adequate for all lifestages. Food intake should not increase during the first 5 weeks of gestation; however,the food intake requirements increase to 1.25 to 1.5 times maintenance during the lasttrimester. During pregnancy in the bitch, protein requirements increase by up to70% compare with maintenance to reach 6.3 g of protein per 419 kJ (100 kcal).1

High-quality, animal-based proteins are preferred. Protein deficiency during preg-nancy can result in low birth weights and high neonatal mortality.2 During lactation,

Nestle Purina PetCare, One Checkerboard Square, St. Louis, MO 63164, USAE-mail address: deborah.greco@rdmo.nestle.com

Vet Clin Small Anim 44 (2014) 265–273http://dx.doi.org/10.1016/j.cvsm.2013.11.001 vetsmall.theclinics.com0195-5616/14/$ – see front matter � 2014 Elsevier Inc. All rights reserved.

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protein requirements are even higher, particularly in large litters.3 Feeding multipletimes a day, with concentrated high-quality food, is essential to maintain body condi-tion of the bitch, otherwise milk production decreases and neonatal growth is affected.In queens, certain amino acids such as taurine play an important role in maintenanceof pregnancy. Fat delivers more kilojoules per gram of food (twice as many) thancarbohydrates or protein, making it an essential component of an energy-rich dietfor late gestation and lactation. However, lactation is extremely demanding andenergy requirements may increase by 3 times compared with basal requirements bythe time of weaning, particularly in large litters. Although protein and fat intake shouldincrease in pregnant and lactating bitches, maintenance of ideal body condition is animportant aspect of feeding pregnant and lactating bitches. Bitches that becomeoverweight have a greater chance of pseudocyesis and dystocia.4

Before birth, some important nutritional requirements can affect the long-termhealth and well-being of puppies. Fats and essential fatty acids, such as linoleicand alpha-linoleic acid, are in increased demand during pregnancy and lactation inthe bitch. Essential fatty acid deficiency has been associated with preterm labor,poor placental development and small litter size.1 In particular, dams fed a diet richin docosahexaenoic acid (DHA) deliver puppies that have improved learning ability,memory, and vision.5–7

Pregnant and lactating bitches do not require calcium supplementation unless tobalance a homemade diet or to treat eclampsia, if present. The proper calcium/phos-phorus ratio in diets for pregnant and lactating bitches is 1:0.8. Normal dietary calciumduring gestation does not increase the risk for eclampsia in dogs; oversupplementa-tion can induce parathyroid gland atrophy and associated postpartum hypocalcemia.1

Concerns have been raised about the role of calcium in the periparturient period.Increased stillbirths have been associated with lower ionized Ca and higher parathy-roid hormone levels in German shepherd bitches.8 During lactation in the bitch, the de-mand for calcium increases exponentially.9,10 In particular, small breed dogs such asChihuahuas may be at increased risk for eclampsia, particularly if they are being fedhomemade diets, such as chicken and rice, without calcium supplementation.Prenatal folate supplementation has been promoted in human medicine to alleviate

midline closure defects such as spina bifida and cleft palate. The only study in dogsshowed that supplementation of folate in Boston terriers resulted in a decrease in thepercentage of puppies with cleft palates.11 However, the study was performed withdogs fed a homemade diet deficient in grains that contain large amounts of folate.11

Prebiotics

Sterile at birth, the GI tract is colonized within hours of birth. This well-controlled colo-nization involves a variety of organisms that find their own niches along the GI tract sothat different groups of microorganisms colonize various locations of the intestinaltract. During the colonization process, these bacteria are organized into a state ofequilibrium (a balance between beneficial and harmful bacteria) so that the health ofthe animal is maintained. In the stomach, because of its oxygen content and acidity,resident bacteria numbers are limited to 103 to 104 bacteria per gram of stomach con-tents. The primary bacteria populating the stomach are lactobacilli and enterococci.Bacteria in the small intestine are a combination of facultative anaerobes and anaer-obes whose growth is limited by peristaltic activity. Populations of these microorgan-isms are low in the upper small intestine, but increase throughout the tract from theduodenum to the ileum. The greatest colonization of the GI tract (approximately200 species or 400 different strains of bacteria) occurs in the colon, where decreasedintestinal transit time allows bacteria to reach numbers of 1011 to 1012 organisms per

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gram. Bacterial flora in the colon is dominated by anaerobes such as intestinal micro-flora (Bacteroides, Eubacterium, Bifidobacterium, Lactobacillus, and Enterococcus).Because the gut microbiota can play a major role in the health of the host animal,

there is significant interest in the manipulation of it toward a potentially more remedialcommunity. Indigestible oligosaccharides, in general, are prebiotic fibers and havebeen shown to modulate the colonic microbiota by increasing the number of so-called good bacteria, thus changing the composition of the microbiota. The character-istics of a beneficial prebiotic are that it passes unchanged through the digestivesystem until it reaches the large intestine; it has metabolic selectivity for good bacteria;it alters microflora balance in favor of a healthier composition; and it induces effectsthat are beneficial to the animal.Prebiotic fibers have been reported to have benefits such as immune stimulation,

anticancer effects, antidiarrhea effects, and increased nutrient absorption. Popularprebiotics include inulin, aleurone, fructo-oligosaccharides (FOSs), mono-oligosaccharides, and beet pulp. Studies have shown that dietary supplementationof short chain fructo oligosaccharides (scFOS) to pregnant and lactating bitchesincreased colostrum and milk immunoglobulin (Ig) M content without a concomitanteffect on IgG1, IgG2, and IgA.2 Furthermore, intranasally immunized puppies showedhigher Bordetella bronchiseptica–specific IgM response.12

PEDIATRIC PUPPIES: WEANING TO 6 MONTHS

Growth rates differ greatly between small and medium, large, and even giant breedpuppies. For example, a miniature poodle may be sexually and physiologically matureat 6 to 8 months of age, whereas a Great Dane may not achieve maturity until 28 to36 months (3 years) of age. As a result of these breed size differences in the rate ofgrowth, food companies have designed foods for small, medium, large, and giantbreeds. The smaller puppy diets are higher in energy and protein, whereas the largeand giant breed diets are lower in caloric content but still high enough in protein toallow proper growth and development. Overfeeding large breed puppies has beenshown to increase the incidence of osteoarthritis (OA) and obesity.13 Mismatch inthe amount of protein and energy in the diet may result in relative protein deficiencythat can cause immune problems and carbohydrate intolerance later in life.14 Puppyfoods that match the amount of protein to the amount of energy in the diet providethe ideal diet for growth in small, large, and giant breed puppies. Ideal foods forgrowing puppies contain at least 25% protein on a percent energy basis. Feeding in-structions should be considered to be guidelines because individual puppiesare different and may require adjustments to keep them in a lean body condition (4–5 out of 9 on the Purina scale). Excess body weight is recognized as a risk factorfor OA in both humans and dogs, whereas avoiding obesity can help reduce OA.Dogs maintained in lean body condition through food restriction experienceddecreased severity, and delayed onset, of OA.13–16

The amounts of omega-3 and omega-6 fatty acids are essential to promoting ahealthy immune system, skin, and hair coat both in utero and in developing puppies.Foods should contain the proper amount (0.05% DHA, Association of American FeedControl Officials [AAFCO] recommendation) of essential fatty acids, including linoleicacid, for growth and development. Feeding diets rich in DHA to puppies up to12 weeks in age improves memory and vision. Omega-3 fatty acids, such as DHA,are required for normal development of retinal function and hearing in puppies.Differences in mineral requirements between large and small breed puppies may be

one of the most important aspects of puppy nutrition. Although miniature poodle

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puppies grow and develop normally on a wide range of calcium intakes, Great Danepuppies require a specific amount (1.5%) of calcium for normal skeletal growth. Skel-etal defects (hip dysplasia, panosteitis, and so forth) and depressed growth wereobserved when high levels of calcium were fed to Great Dane puppies; normal growthand development were observed when the puppies were fed 0.80% to 1.5 % Ca on aDMB.17 Severely reduced growth and pathologic fractures were noted in Great Danepuppies fed less than or equal to 0.55% Ca on a DMB (low calcium).17 Small, large,and giant breed puppy foods have varying amounts of calcium and phosphorus inthe proper ratios to control growth and development of bones and cartilage. The adultsize of a puppy is not determined by the rate of growth (accelerated beyond normal),but by the genetic makeup of the puppy (the size of the parents). Again, slower, morecontrolled growth of the skeleton is associated with more normal development. Rapidgrowth has been associated with the development of OA, osteochondrosis dissecans,hip dysplasia, and metabolic bone disease in large and giant breed dogs.15,17 Calciumsupplements should not be used because of the detrimental effects on bone growth,particularly in large breed puppies. Natural supplements, such as yogurt and cottagecheese, contain excessive amounts of calcium; for example, yogurt contains 450 mgof Ca per cup.

SUPPLEMENTS THAT AFFECT THE IMMUNE SYSTEM IN PUPPIES AND KITTENS

The gut is the largest immune system organ in the body. About 70% of the body’simmune system is located in the GI tract. As such, the interaction between the gut-associated lymphoid tissue and microflora is key to overall health. The most importantimmune-related function of the good intestinal microflora is protection against infec-tion and colonization by harmful, and sometimes pathogenic, bacteria. This protectionis provided by physical barriers and resistance to pathogen colonization. Both of theseprotective mechanisms can be altered by several factors. Aging and stress associatedwith traveling, kennel boarding, changes in environment or diet, and poor nutrition alsocan affect the balance. One study that investigated the effects of aging on the intes-tinal microflora of dogs found that young adult dogs had higher levels of lactobacilliand lower levels of Clostridium perfringens compared with elderly dogs.1 Stress cantrigger changes in intestinal pH, contractility, and transit time, resulting in an imbal-ance in microflora. Shifts in microbial populations can result in increased sheddingof pathogenic bacteria and reduced performance of the protective barrier of thegut, which can lead to GI upset and systemic problems. Even otherwise goodstresses, such as traveling for a competitive dog event, can upset this delicatebalance. Two recent advances in veterinary nutrition, bovine colostrum supplementa-tion and probiotic therapy, may help prevent some of the digestive upset associatedwith unavoidable stressors such as weaning and travel.

Colostrum

As nutritional science has become more advanced, certain nutrients and ingredientshave been identified that provide more benefit to the animal than was previouslythought. Colostrum is one of these entities that provides more benefit than was previ-ously thought. Neonatal mammals acquire passive immunity during the first 24 hoursof life because a permeable GI tract allows large immunoglobulins in the first mother’smilk to pass freely through the intestinal barrier for about 24 to 48 hours after birth.However, it seems that kittens and puppies may have a shorter window for transferof factors, with optimum transfer in the first 3 to 6 hours and completion by 16 to24 hours.18,19 These nutrients and ingredients contain bioactive compounds that

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positively influence both the immune and GI systems. Bovine colostrum has beenidentified as one such immunostimulatory bioactive ingredient. Recent research hasshown that natural antibodies (also called immunoglobulins) and other bioactivegrowth factors found in colostrum can help strengthen the immature immune systemof a puppy or kitten. It can help balance the beneficial and harmful bacteria in theintestine, which not only promotes nutrient absorption facilitated by colonic microflorabut lowers the potential for infection, diarrhea, and intestinal inflammation. In addition,it is a good source of readily available amino acids for gut cell health and barrierfunction.Although the neonate is born with a functional immune system, it is still immature

and naive at birth. Its response to any immunostimulation is that of a first exposure,requiring a prolonged period to produce immunoglobulins. Colostrum provides immu-noglobulins and other bioactive growth factors that stimulate development of the GItract.The importance of colostrum in helping establish a healthy intestinal microflora pop-

ulation has been investigated.20–22 Human studies show that breastfed infants have apredominance of beneficial bacteria (bifidobacteria and lactobacilli) and lower fecalpH compared with those fed a pasteurized cow milk–based formulation. The endog-enous intestinal bacteria compete with pathogens and provide an environment thatfavors beneficial bacteria. In addition to the physical barrier provided by the mucousmembranes lining the GI tract and the immunologic power of gut-associated lymphoidtissue, the intestinal microflora have an important role as part of the body’s naturaldefense system.New research has been conducted to investigate the use of colostrum to enhance

immunity and modulate gut microflora in both kittens and puppies.22 Colostrum is acombination of secretions from the mammary gland and proteins transferred fromthe blood stream. Many studies suggest that various constituents of milk are bioactivebecause of their positive health effects. For example, bovine milk components repre-sent a potentially useful way to modulate gut microflora and immunity. Bovine colos-trum and the whey fraction (a protein-rich complex derived from bovine milk) containnot only immunoglobulins but also lactoferrin, lactoglobulin, lactalbumin, all of whichhave been shown to modulate immune function as well as intestinal microflora. Lacto-ferrin inhibits undesirable microflora by sequestering iron in the gut, and promotes thegrowth of the beneficial bacteria, lactobacilli and bifidobacteria. Glycomacropeptide isknown to regulate appetite as well as act as a prebiotic and immunostimulant. Cyto-kines and growth factors such as epidermal growth factor, tumor growth factor alpha,tumor growth factor R, insulinlike growth factors and binding proteins, platelet-derivedgrowth factor, vascular endothelial growth factor, and growth hormone play importantroles in maintaining a healthy gut wall.Immunoglobulins are Y-shaped molecules in which the shorter arm of the Y is

named the fragment antigen binding (Fab) region and the longer body is named thefragment crystalline (Fc) region. The Fab region binds specific antigens, whereasthe Fc region is responsible for most of the biologic activity by interacting with theimmune cells. Most of the immunoglobulins introduced into the gut remain intactthrough the stomach into the small intestine where the immune cells reside. Slightdenaturing may enhance the ability of the Fc region to bind immune cells and stimulatethe immune system. When colostrum is delivered with a complete and balanced dietthere is ample foodmatrix available for degradation by the stomach enzymes, which inturn causes decreased pH. Immunoglobulins are small and therefore not a high priorityfor processing by the stomach before entering the small intestines. Based on studiesin other species, the relevant components of the immunoglobulins remain intact

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through digestion and have a positive immunomodulating effect on digestion. As aresult, most immunoglobulins in the diet reach the small intestine, where they bindand stimulate immune cells and boost the immune status of the animal. Therefore,the ultimate benefits of immunoglobulins are their ability to bind the gut immune cells,which in turn stimulates the local and systemic immune system and results in overallenhanced immunity.One of the protective functions of the gut is to eliminate pathogenic bacteria and

neutralize toxins. Bioactives in colostrum have been shown to bind specific receptorson the gut immune cells and activate them to secrete higher levels of IgA into thelumen of the gut. Secretory IgA prevents adherence of pathogens to mucosal sur-faces, transports potential pathogens out of the cells into the lumen, binds virus,and prevents replication. The gut IgA is eventually excreted in the feces. Therefore,a higher level of fecal IgA is desirable because it indicates stimulation of the localmucosal immune system. Colostrum stimulates the gut immune system to secretehigher levels of secretory IgA.Bacterial populations are in constant flux in the gut because of cellular turnover,

introduction of food, and interaction of the gut microenvironment. Puppies and kittensfed the colostrum diet had more similarity in their microbial population before and afterstress. A more stable microbial population leads to optimized nutrient absorption anddecreased susceptibility to stress-induced or stress-related diarrhea.A complete and balanced diet supplemented with colostrum enhanced the

systemic immune status and digestive health of puppies and kittens without causingoverstimulation of the immune system. In addition, the puppies and kittens fed colos-trum had higher fecal IgA, indicating a higher local gut immune response. Increasedsimilarity of bacterial populations in the gut before and after stress, natural antibodies,and other bioactives in colostrum help to balance beneficial and potentially harmfulintestinal bacteria. By stabilizing gut microflora, colostrum helps promote nutrient ab-sorption and lowers the potential for infection, diarrhea, and intestinal inflammation.

Probiotics

Enterococci are typical lactic acid bacteria and occur naturally in a wide variety ofenvironments, including certain foods, as well as in the intestinal tract of animals.Much human research has been conducted on the potential benefits of SF68 in pa-tients with acute diarrhea, traveler’s diarrhea, dysbiosis associated with antibioticuse, irritable bowel syndrome, hepatic disorders, and encephalopathy. Probioticshave been shown to decrease the incidence, severity, and duration of diarrhea.In selecting a potential strain to be used as a probiotic for a pet food supplement,

several important factors should be considered, including a long history of safe usein both humans and animals, and whether it has been approved by AAFCO. The po-tential benefits of SF68, based on a large number of trials in humans and animals, wereconsistent with desirable benefits in pets. Probiotics have been shown to survive theconditions of the GI tract in pets based on well-controlled in vitro and in vivo tests.Certain probiotics (such as FortiFlora; Nestle Purina, Vevey, Switzerland) incorporatea proprietary microencapsulation process that allow for stability of the microorgan-isms through the production, distribution, and storage processes.Probiotics have been used successfully in both dogs and cats since 1985 for the

maintenance and restoration of healthy gut microflora in animals showing distur-bances caused by changes in diet, stress, and antibiotic therapy. Enterococcus fae-cium can temporarily survive in the GI tract of healthy adult dogs for about 14 days.Feeding E faecium reduced the fecal concentration of C perfringens in dogs and

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increased levels of fecal bifidobacteria and lactobacilli. Probiotics caused statisticallysignificant improvement in fecal scores in dogs with naturally occurring diarrhea.23

Puppies and kittens can be susceptible to diarrhea, particularly at the time of wean-ing or if insufficient colostrum has been ingested. In both cases, administration of pro-biotics, like E faecium, repopulates the intestine with helpful bacteria for 14 days toallow repair and stabilization of gut flora. Other probiotics are available from severaldifferent pharmaceutical companies; however, most of them have not undergonerigorous clinical trials and studies to prove efficacy. Yogurt contains too much calciumto be used as a probiotic and most yogurts do not contain sufficient measurableamounts of beneficial bacteria to be considered probiotics. The manufacturer shouldbe able to provide documentation as to whether the probiotic has stability (ie, it doesnot revert to a pathogenic strain), and whether it survives in the gut for a least 14 days.The microflora in the GI tract consists of normal and pathogenic microflora. The

helpful microorganisms contribute to intestinal mucosal integrity, metabolism, and im-mune status (both local and systemic). In addition, probiotics or live active cultureshave been shown to have beneficial effects in the host animal by improving its intes-tinal microbial balance. However, one of the most interesting studies on the effects ofE faecium found that there were statistically significant increases in IgA observed inanimals fed the probiotic.22 In cats, decreased recurrence of clinical signs of herpesvirus during stress was also observed when the cats were supplemented with probi-otics.23 This boosting of the immune system in puppies and kittens has important im-plications for improving the health of pets and the immune response to vaccination.

MILK REPLACERS FOR ORPHANED PUPPIES AND KITTENS

The composition of bitch and queen milk compared with commercially available milkreplacers is shown in Table 1. Although there is not much literature pertaining to milkreplacement therapy in orphaned puppies and kittens, a few studies could befound.24,25 The use of a commercially available puppy milk replacer in Denmark wasassociated with overfeeding of puppies; furthermore, the milk replacer had 7-foldthe recommend content of vitamin D.24 The investigators concluded that this couldresult in abnormal skeletal growth and developmental orthopedic problems later inlife. There were similar findings of increased weight gain in kittens fed a commercialmilk replacer (CMR) compared with kittens fed queen’s milk.25 In addition, kittensfed the CMR had diarrhea throughmost of the trial period andmost of them developeddiffuse anterior and posterior lens opacification.25 Serum arginine was significantly

Table 1Comparison of the macronutrient content (percent dry matter [DM]) of commercial milkreplacers to species-specific milk composition

Type Protein (% DM) Fat (% DM) Carbohydrates (% DM)

Bitch milk 33 41 17

Queen milk 42 25 26

Commercial goat milk (Esbilac) 35 42 15

Veterinary dog (Nutri-Cala) 34 43 16

Commercial dog (Just Bornb) 30 30 30

Commercial cat (KMR) 44 26 24

a Contains FOS, colostrums.b Contains colostrum, and arginine to prevent cataracts.

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lower in the CMR-fed kittens and may have contributed to the cataract formation.Based on these findings, currently available milk replacers are often supplementedwith arginine and more restricted in vitamin D supplementation than previously avail-able preparations. Clients should be instructed to follow manufacturer’s feeding in-structions carefully and not to overfeed.

SUMMARY

Feeding a balanced commercial diet to lactating and growing puppies and kittensprovides the necessary macronutrients (protein, fat, and so forth), vitamins, minerals,and supplements required for normal growth and development. Added benefit mayarise from the use of DHA-supplemented diets and from the use of prebiotic fibers,colostrum, and probiotics to promote growth and development of a healthy GI tract,microbiota, and immune system in puppies and kittens.

REFERENCES

1. Kirk CA. New concepts in pediatric nutrition [review]. Vet Clin North Am SmallAnim Pract 2001;31(2):369–92.

2. Ontko JA, Phillips PH. Reproduction and lactation studies with bitches fed semi-purified diets. J Nutr 1958;65:211–8.

3. Schroeder GE, Smith GA. Bodyweight and feed intake of German shepherdbitches during pregnancy and lactation. J Small Anim Pract 1995;36(1):7–11.

4. Lawler DF, Johnston SD, Keltner DG, et al. Influence of restricted food intakeon estrous cycles and pseudopregnancies in dogs. Am J Vet Res 1999;60(7):820–5.

5. Heinemann KM, Waldron MK, Bigley KE, et al. Long-chain (n-3) polyunsaturatedfatty acids are more efficient than alpha-linolenic acid in improving electroretino-gram responses of puppies exposed during gestation, lactation, and weaning.J Nutr 2005;135(8):1960–6.

6. Heinemann KM, Bauer JE. Docosahexaenoic acid and neurologic developmentin animals [review]. J Am Vet Med Assoc 2006;228(5):700–5, 655.

7. Bauer JE, Heinemann KM, Bigley KE, et al. Maternal diet alpha-linolenic acid dur-ing gestation and lactation does not increase docosahexaenoic acid in caninemilk. J Nutr 2004;134(Suppl 8):2035S–8S.

8. Hollingshead. Calcium and parathormone concentrations in German shepherdbitches during labor (abstract). Proc World Sm Anim Vet Asso. 2007.

9. Adkins Y, Lepine AJ, Lonnerdal B. Changes in protein and nutrient composition ofmilk throughout lactation in dogs. Am J Vet Res 2001;62(8):1266–72.

10. Lawler DF. The role of perinatal care in development [review]. Semin Vet MedSurg (Small Anim) 1995;10(1):59–67.

11. Elwood JM, Colquhoun TA. Observations on the prevention of cleft palate in dogsby folic acid and the potential relevance to humans. N Z Vet J 1997;45(6):254–6.

12. Adogony V, Respondek F, Biourge V, et al. Effects of dietary scFOS on immuno-globulins in colostrum and milk of bitches. J Anim Physiol Anim Nutr (Berl) 2007;91:169–74.

13. Dobenecker B, Endres V, Kienzle E. Energy requirements of puppies of twodifferent breeds for ideal growth from weaning to 28 weeks of age. J Anim PhysiolAnim Nutr (Berl) 2013;97:190–6.

14. Kealy RD, Lawler DF, Ballam JM, et al. Effects of diet restriction on life span andage-related changes in dogs. J Am Vet Med Assoc 2002;220(9):1315–20.

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15. Nap RC, Hazewinkel HA, Voorhout G, et al. Growth and skeletal development inGreat Dane pups fed different levels of protein intake. J Nutr 1991;121(Suppl 11):S107–13.

16. Kealy RD, Lawler DF, Ballam JM, et al. Evaluation of the effect of limited food con-sumption on radiographic evidence of osteoarthritis in dogs. J Am Vet Med Assoc2000;217(11):1678–80.

17. Schoenmakers I, Hazewinkel HA, Voorhout G, et al. Effects of diets with differentcalcium and phosphorus contents on the skeletal development of growing GreatDanes. Vet Rec 2000;147(23):652–60.

18. Chastant-Maillard S, Freyburger L, Marcheteau E, et al. Timing of the intestinalbarrier closure in puppies. Reprod Domest Anim 2012;47(Suppl 6):190–3.

19. Casal ML, Jezyk PF, Giger U. Transfer of colostral antibodies from queens to theirkittens. Am J Vet Res 1996;57(11):1653–8.

20. Giffard CJ, Seino MM, Markwell PJ, et al. Benefits of bovine colostrum on fecalquality in recently weaned puppies. J Nutr 2004;134:2126S–7S.

21. Satyaraj E, Reynolds A, Pelker R, et al. Supplementation of diets with bovinecolostrum influences immune function in dogs. Br J Nutr 2013;1–6.

22. Benyacoub J, Czarnecki-Maulden GL, Cavadini C, et al. Supplementation of foodwith Enterococcus faecium (SF68) stimulates immune functions in young dogs.J Nutr 2003;133(4):1158–62.

23. Lappin MR, Veir JK, Satyaraj E, et al. Pilot study to evaluate the effect of oral sup-plementation of Enterococcus faecium SF68 on cats with latent feline herpes virus1. J Feline Med Surg 2009;11:650.

24. Corbee RJ, Tryfonidou MA, Beckers IP, et al. Composition and use of puppy milkreplacers in German Shepherd puppies in the Netherlands. J Anim Physiol AnimNutr (Berl) 2012;96:395–402.

25. Remillard RL, Pickett JP, Thatcher CD, et al. Comparison of kittens fed queen’smilk with those fed milk replacers. Am J Vet Res 1993;54:901–7.