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animals
Article
Comparison of Rabbit, Kitten and Mammal Milk
Replacer Efficiencies in Early Weaning Rabbits
Panthiphaporn Chankuang 1, Achira Linlawan 1, Kawisara Junda 1, Chittikan Kuditthalerd 1,
Tuksaorn Suwanprateep 1, Attawit Kovitvadhi 2, * , Pipatpong Chundang 2,
Pornchai Sanyathitiseree 3and Chaowaphan Yinharnmingmongkol 4
1Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand;
panthiphaporn.c@ku.th (P.C.); achira.l@ku.th (A.L.); kawisara.ju@ku.th (K.J.); chittikan.k@ku.th (C.K.);
tuksaorn.s@ku.th (T.S.)
2Department of Physiology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand;
pichandang@gmail.com
3Department of Large Animal and Wildlife Clinical Science, Faculty of Veterinary Medicine,
Kasetsart University, Nakorn Pathom 73140, Thailand; fvetpos@ku.ac.th
4Animal Space Pet Hospital, Bangkok 10170, Thailand; taaoy@hotmail.com
*Correspondence: fvetawk@ku.ac.th; Tel.: +66-89-2022-677
Received: 19 May 2020; Accepted: 20 June 2020; Published: 23 June 2020
Simple Summary:
A milk replacer must be given as the main diet to young rabbits that are separated
from their mothers before they reach weaning age (31–35 days). This procedure, which is a rescue
protocol, allows them to survive. Moreover, the early separation of young rabbits before weaning
prevents negative consequences in lactating rabbits, which is beneficial to pet rabbit producers.
Kitten (KMR
®
, Pet-Ag Inc., Hampshire, IL, USA: KMR) or mammal (Zoologic
®
Milk matrix 30/52,
Pet-Ag Inc., Hampshire, IL, USA: MMR) milk replacers have generally been suggested for use in
rabbits; however, rabbit milk has a unique composition. Therefore, a rabbit milk replacer (RMR) was
formulated in this study for comparison with these commercial products. Early weaned rabbits at
18 days of age were fed daily with RMR, KMR or MMR until 36 days after birth, while a commercial
pelleted diet and water were provided at an amount exceeding the normal intake. The results indicated
that it is possible to use RMR as a milk replacer for rabbits without serious adverse consequences.
However, the RMR group presented a lower trend in nutrient digestibility than the other groups,
although there was no statistical significant difference. Therefore, prebiotics and/or probiotics should
be added to RMR formulations to improve this parameter.
Abstract:
Early weaned rabbits should be fed using a milk replacer in order to survive. Therefore, a
rabbit milk replacer (RMR) was developed and compared with a kitten milk replacer (KMR
®
: KMR)
and a mammal milk replacer (Zoologic
®
Milk matrix 30/52: MMR). Thirty-six native crossbred rabbits
aged 18 days were divided into three experimental groups (six replicates/group, two rabbits/replicate),
fed RMR, KMR or MMR daily until they were 36 days old and euthanized at 38 days, while a complete
pelleted diet and water were provided ad libitum. No statistically significant differences were observed
in growth performance parameters, water intake, faecal weight, nutrient digestibility, internal organ
weight, caecal pH, caecal cellulose activity, number of faecal pellets and amount of crude protein
intake (p>0.05). Caecal amylase activity in the KMR group and caecal protease activity in the RMR
group were higher than in the MMR group (p<0.05). The villus height and crypt depth of the MMR
group were greater than in the RMR and KMR group (p<0.05). In conclusion, it is possible to feed
RMR to early weaning rabbits without serious adverse effects. However, probiotics and/or prebiotics
should be supplemented in milk replacers and their benefits studied.
Keywords: digestibility; enzyme activity; gut histology; milk replacer; rabbit
Animals 2020,10, 1087; doi:10.3390/ani10061087 www.mdpi.com/journal/animals
Animals 2020,10, 1087 2 of 12
1. Introduction
The size of the pet market has increased sharply in recent years and was estimated to be around
131.7 billion US dollars in 2016 [
1
]. Moreover, the compounded annual growth rate of the global pet care
market was forecast to be 4.9% between 2018 and 2025 due to changes in the new generation’s lifestyle,
such as living alone or child-free marriage [
1
]. Nevertheless, humans still need interactions with
living things, which add social, medical, emotional and physical benefits to their lives [
2
]. Companion
animals are one solution that can offer these benefits [
2
]. Although rabbits are not as popular as dogs
and cats, they occupy third place among companion animals because they are clean, quiet, non-harmful
and require little space [3].
Generally, rabbits are weaned at 31–35 days of age by rabbit producers in Thailand and other
countries [
4
,
5
] and begin to consume pelleted diets around 18 days of age [
6
]. Milk replacer has been
suggested as a means of feeding early weaned rabbits and orphaned rabbits and of solving the problem
of female rabbits that do not produce milk, this being rabbits’ major nutrient source for survival [
7
] as
well as preventing gastrointestinal disease [
8
]. The early separation of young rabbits from their mother
can prevent a negative energy balance due to lactation, which supports a higher production yield and
reduces disease transmission from the mother to young rabbits, of benefit for pet rabbit producers [
4
,
9
].
In addition, the smallest rabbit breed (Netherland Dwarf) can produce around 4–6 kittens per litter;
however, the milk yield of this breed is not sufficient to support their kittens, leading to a high mortality
rate among young rabbits [
5
]. Furthermore, milk replacer can be used to rescue unweaned wild
rabbits [8]. Therefore, the use of a milk replacer provides one solution to these problems.
Commercial rabbit milk replacer remains lacking or unavailable in some countries. For this
reason, kitten milk replacer (KMR) has been suggested as a substitute [
7
]. However, although kitten
milk replacer can be used in early weaning rabbits, its growth performance has been found to be
inferior to rabbit milk for rabbits [
8
]. In addition, two milk replacer formulas using mixtures of
kitten milk replacers—Fox Valley Ultraboost and/or Fox Valley 32/40 (Fox Valley Animal Nutrition,
INC., Lakemoor, IL, USA)—have been used to rescue young desert cottontail (Sylvilagus audubonii)
and eastern cottontail rabbits (S. floridanus), for which the mortality rate was 26–59% [
8
]. The high
concentration of nutrients (fat, protein and energy), the near absence of lactose, the high proportion
of medium-chain saturated fatty acids with bacteriostatic properties (C8:0 and C10:0) and the short
milking period required constitute the unique characteristics of rabbit milk and feeding behaviour [
6
,
10
].
Therefore, milk replacer for rabbits should be formulated respecting the properties of real rabbit
milk [
10
]. Moreover, cost-effectiveness is another problem for rabbit producers, owners and wildlife
rescue center [
8
]. Therefore, this study aimed to compare the efficiency of a developed rabbit milk
replacer with two commercial products (kitten and mammal milk replacers) based on the growth
performance and health status of early weaning rabbits (18 days old).
2. Materials and Methods
2.1. Ethics Statement
This study was conducted following standard guidelines at the animal experimental unit, Faculty
of Veterinary Medicine (Kasetsart University, Bangkok, Thailand) and was approved by the Institutional
Animal Care and Use Committee of Kasetsart University, Bangkok, Thailand (ACKU62-VET-037).
2.2. Animals, Diets, Milk Replacer Preparation and Experimental Design
Thirty-six 18-day-old native crossbreed rabbits with initial body weights of 134
±
6.31 g/head
(mean
±
standard deviation) were taken from a local rabbit farm (Saha farm, Kanchanaburi, Thailand).
Rabbits were randomly separated into three experimental groups with equal numbers of each sex
(six replicates per group and two rabbits per replicate) containing: (1) rabbits fed rabbit milk replacer,
which was formulated in this study (RMR); (2) rabbits fed kitten milk replacer (KMR
®
; Pet-Ag Inc.,
Hampshire, IL, USA); and (3) rabbits fed mammal milk replacer (Zoologic
®
Milk matrix 30/52; Pet-Ag
Animals 2020,10, 1087 3 of 12
Inc., Hampshire, IL, USA; MMR). Rabbits were placed in a stainless cage (35 cm
×
35 cm
×
35 cm)
with controlled room temperature, light and humidity at 20
±
2
◦
C, 16L:8D and 75
±
10%, respectively.
The experiment was conducted for 20 days until the rabbits reached 38 days of age. At the end of the
experiments, one rabbit per replicate was euthanized by intraperitoneal injection with pentobarbital
sodium at 100 mg/kg (Nembutal, Ceva corporate, France) [
11
] and the samples were collected for
further analysis, while another rabbit of each replicate was returned to the farm and reared until it
reached 60 days of age.
The formulation of the RMR, including the chemical composition of the milk replacer, complete
pelleted diet and rabbit milk, is illustrated in Table 1. Every day, rabbits were fed 10 mL at 38
◦
C of a
freshly prepared mixture of milk replacer powder and clean water using a sterile syringe at 7:00 until
they reached weaning age (36 days), while clean water and complete commercial rabbit diets (Lee Feed
Mill, Publ. Co., Ltd., Phetchaburi, Thailand) were provided ad libitum throughout the experiment.
The KMR and MMR powders were diluted with warmed water at a 7:13 ratio and homogenized.
The RMR consisted of two parts: a hydrogenated palm fat part and a mixed dried powder part,
containing all ingredients except hydrogenated palm fat and polyoxyethylene (80) sorbitan monooleate.
For RMR preparation, hydrogenated palm fat was heated in an 800 W microwave for 60 s, changing
from solid to liquid form as a result. The dried mixed powder part was then mixed with warm water.
Subsequently, the two parts were homogenized and polyoxyethylene (80) sorbitan monooleate was
added as an emulsifier. The ratio of mixed dried powder to hydrogenated palm fat to warm water
was 24.5:17.5:78. The dilution ratio was selected based on equal dry matter content between the milk
replacers and the solubility of the milk mixture.
Table 1. Ingredients and chemical components of different milk replacers, rabbit milk and diet.
Items
Artificial Milk Replacers
Rabbit Diet aRabbit Milk b
RMR KMR MMR
Ingredients (%)
Sodium caseinate 43.8 - - - -
Skimmed milk powder 7.73 - - - -
Hydrogenated palm fat 41.5 - - - -
Monodicalcium phosphate 3.13 - - - -
Limestone 2.30 - - - -
Salt 0.94 - - - -
Premix c0.50 - - - -
Polyoxyethylene (80) sorbitan monooleate 0.10 - - - -
Chemical composition
Dry matter (%FM) 95.8 96.2 95.2 90.7 29.8
Crude ash (%DM) 9.77 6.66 8.27 5.69 7.38
Crude protein (%DM) 41.4 48.5 30.1 16.1 41.3
Ether extract (%DM) 43.8 22.9 52.5 2.42 43.3
Crude fiber (%DM) ND ND ND 25.2 ND
Nitrogen free extract (%DM) d5.03 22.0 9.13 50.6 8.05
Metabolizable energy content (kcal/100g DM) e535 441 584 254 540
RMR =Rabbit milk replacer which was performed in this study, KMR =Kitten milk replacer (KMR
®
, Pet-Ag Inc.,
Hampshire, IL, USA), MMR =Mammal milk replacer (Zoologic
®
Milk matrix 30/52, Pet-Ag Inc., Hampshire, IL,
USA), FM =Fresh matter, DM =Dry matter, ND =Not detect;
a
A commercial pelleted diet for rabbits (Lee Feed Mill,
Publ. Co., Ltd., Phetchaburi, Thailand);
b
Chemical composition of rabbit milk [
10
];
c
Vitamin and mineral premix
(Topmix-B111, Top Feed Mills Co., Ltd., Pathumthani, Thailand) were supplied per kilogram of diets at 4,800,000 IU
of vitamin A; 1,200,000 IU of vitamin D3; 6000 IU of vitamin E; 600 mg of vitamin K; 600 mg of vitamin B1; 2200 mg
of vitamin B2; 10,000 mg of vitamin B3; 800 mg of vitamin B6; 4 mg of vitamin B12; 48 mg of biotin; 4800 mg of
Calcium pantothenate acid; 200 mg of folic acid; 24,000 mg of Zn, 16,000 mg of Fe; 32,000 mg of Mn; 32,000 mg of
Cu; 200 mg of I; 40 mg of Se; 40 mg of Co; dCalculation [12]; eCalculation based on Atwater system [13].
2.3. Perfomance, Digestibility and Faecal Evaluation
The animals were weighed at 18, 24, 30 and 36 days of age, whereas average daily feed intake
(ADFI), average daily weight gain (ADG), feed conversion ratio (FCR), water intake and weight of
faeces output were evaluated at 19–24, 25–30 and 31–36 days of age. The apparent digestibility of dry
matter, organic matter, ether extract and crude protein was conducted at 23–27 and 31–35 days of age
Animals 2020,10, 1087 4 of 12
and contained six replicates/groups. The procedures for feeding, faecal collection, chemical analysis
and calculation were in accordance with [
14
]. Briefly, feed intake was measured during the period of
the digestibility trial. Faeces were removed from the cage at 9:00 on the first day of the digestibility
trial. Subsequently, all faeces on a net under the cage were collected at 9:00 for four days. The faeces
were weighted immediately after collection, put in a sterile plastic bag and kept at −20 ◦C for further
chemical composition analysis following the procedure of [
14
]. Another study was conducted, where
the amount of daily faecal pellet excretion was measured by counting the dried faecal pellets between
19 and 36 days old from photos.
2.4. Internal Organs, Gut Histology and Caecal pH
The internal organ weight and the body weight of the euthanized rabbits were determined.
The duodenal part of the small intestine was fixed in 10% buffered formalin for further villus
morphometric evaluation. Briefly, small pieces of middle duodenum after fixation were processed,
embedded in paraffin, sectioned at 7-
µ
m thicknesses by means of a rotary microtome (Leica RM2155;
Leica Instruments GmbH, Nussloch, Germany) and stained by haematoxylin and eosin method.
Villi height and crypt depth were evaluated under a microscope using an image analysis programme
(Image Pro Plus; Media Cybernetics, Bethesda, MD, USA). Caecal pH was measured directly using
a Crison MicropH 2001 pH meter (Crison Instruments, Barcelona, Spain). The caecal content was
immediately placed in sterile plastic tubes under ice for enzyme preservation and kept at
−
20
◦
C for
further analysis of caecal enzyme activity.
2.5. Caecal Enzyme Activity
The crude enzyme extracted from the caecal content was extracted by homogenized caecal content
with phosphate buffer solution (pH 7) at a 1:5 ratio (w/v). The homogenates were centrifuged at
18,000
×
gfor 30 min at 4
◦
C to obtain the supernatant used to evaluate amylase, protease and cellulase
activity. Amylase and cellulase activity were assayed according to [
15
,
16
] using 5% soluble starch and
1% carboxyl-methyl cellulose (CMC; medium viscosity) as the substrate, respectively. One hundred
microlitres of crude enzyme extract were added to activate the digestion of the substrates. The products
of the carbohydrate-digestive enzymes were stained using 1% dinitrosalicylic acid (DNS) and measured
using a spectrophotometer at 540 nm against a linear range of maltose standards for amylase and
glucose standards for cellulase. Protease activity was assayed according to the method described
by [
17
] using 0.6% casein as the substrate. The product of the protein-digesting enzyme was measured
spectrophotometrically at 660 nm against a linear range of tyrosine. The activity of the observed
digestive enzymes was expressed as U.
2.6. Crude Protein Assessment
Each rabbit’s feed intake between 19–24, 25–30 and 31–36 days old and the amount of crude protein
in the milk replacer and diet were used as information to calculate the amount of crude protein intake.
2.7. Statistical Analysis
The results of this study are represented as the mean and pooled standard error of the mean.
A completely randomized design was employed in this study. Therefore, one-way analysis of variance
(ANOVA) was used to compare the different types of milk replacers (fixed factors) for internal organ
characteristics, caecal pH, caecal digestive enzyme activities and duodenal histology, whereas the
growth performances, water intake, faeces excretion, apparent digestibility, number of faecal pellets
and amount of protein intake were analyzed by two-way mixed analysis of variance, with treatment
groups or age serving as the between-subjects or the within-subjects factor, respectively. Duncan’s
multiple range test was used for post hoc analysis. Differences were considered statistically significant
at p<0.05. All statistical analyses in this study were performed with R-statistic software using the
Rcmdr package [18].
Animals 2020,10, 1087 5 of 12
3. Results
The effects on performance, apparent digestibility, amount of faeces excretion and crude protein
intake from rabbits fed the different milk replacers are shown in Table 2. No statistically significant
differences between the groups and the interactions between the studied factors (groups and age) for all
parameters in Table 2were apparent (p>0.05). The age increment was correlated with increased body
weight, ADFI, ADG, FCR, water intake, faeces excretion and crude protein intake (p<0.05), whereas
apparent digestibility did not affect dry matter, organic matter or ether extract (p>0.05). However,
the crude protein digestibility of rabbits at 31–35 days old was lower than rabbits at
23–27 days
old
(p<0.05). The rabbits fed KMR and MMR displayed higher nutrient digestibility in both age ranges
compared with rabbits fed RMR, but there was no statistically significant difference (p>0.05). Rabbits
in RMR, KMR and MMR were received the crude protein from milk daily at 2.23, 2.61 and 1.62 g dry
matter/head. No deaths, morbidities or clinical signs were observed in rabbits during the experimental
period. Moreover, a rabbit in each replicate was not euthanized at the end of the experiment and
remained alive until it reached two months of age. In addition, there were no problems of milk
perception and palatability in any group in this experiment, because the rabbits sucked milk directly
from the syringe without any force feeding.
Table 2.
Effect of different milk replacers on rabbit performances, apparent digestibility and crude
protein intake.
Parameters
Factors
SEM
p-Value
Artificial Milk Replacers (AMR) Age (days) AMR Age AMR * Age
RMR KMR MMR 0 6 12 18
BW (g/head) 210 198 202 134 a157 b214 c308 d9.086 0.38 0.001 0.94
19–24 25–30 31–36 -
ADFI (g/head/day) 22.1 26.7 22.1 7.47 a22.1 b39.6 c- 2.239 0.59 0.001 0.56
ADG (g/day) 9.62 9.33 8.52 4.40 a10.8 b14.6 c- 0.808 0.84 0.001 0.77
FCR 2.39 2.62 2.74 1.91 a2.21 a2.75 b- 0.115 0.44 0.01 0.56
Water intake
(g/head/day) 41.8 30.9 34.2 10.1 a31.9 b68.0 c- 3.902 0.1 0.001 0.38
Faeces excretion
(g/head/day) 15 14.3 15.4 1.29 a5.49 b8.14 c- 0.936 0.81 0.001 0.32
Crude protein intake (g/head/day) 1
Diet 3.72 3.73 3.9 1.20 a3.56 b6.37 c- 0.361 0.59 0.001 0.56
Diet and milk 5.95 6.34 5.52 3.39 a5.65 b8.56 c- 0.364 0.11 0.001 0.56
23–27 31–35 - -
Apparent digestibility (%)
Dry matter 59.6 63.3 63.5 60.5 63.4 - - 1.126 0.29 0.22 0.96
Organic matter 60.7 65.1 65.2 62.8 64.2 - - 1.123 0.18 0.51 0.85
Ether extract 68.3 73.8 70.7 71.3 70.3 - - 2.418 0.75 0.88 0.97
Crude protein 76.5 80.8 81.1 81.5 b77.1 a- - 1.007 0.07 0.03 0.34
RMR =Rabbit milk replacer, which was used in this study, KMR =Kitten milk replacer (KMR
®
, Pet-Ag Inc.,
Hampshire, IL, USA), MMR =Mammal milk replacer (Zoologic
®
Milk matrix 30/52, Pet-Ag Inc., Hampshire, IL,
USA), SEM =pooled standard error of mean, BW =Body weight, ADFI =Average daily feed intake, ADG =Average
daily weight gain, FCR =Feed conversion ratio;
a, b, c, d
The differences in superscript letter in the same row
represented statistical significant differences (p<0.05);
1
The rabbits in the RMR, KMR and MMR groups received
crude protein from milk at 2.23, 2.61 and 1.62 g/head/day, respectively.
The consequences for internal organ weight, caecal pH, duodenal wall histology and digestive
enzyme activities between the groups are compared in Table 3. The weight of each internal organ was
not statistically significantly different between the groups (p>0.05). Caecal pH was not influenced by
the differences in milk replacers (p>0.05). Caecal amylase activity in the MMR group was lower than
in the KMR group (p<0.05), whereas greater caecal protease activity was observed in the RMR group
compared to the MMR group (p<0.05). Cellulase activity was not affected by the treatments (p>0.05).
Respectively, the shortest villus and the shallowest crypt depth were found in the RMR and the KMR
groups compared to the MMR group (p<0.05).
Animals 2020,10, 1087 6 of 12
Table 3.
Effect of different milk replacers on internal organ weight, caecal pH, intestinal villi morphology
and digestive enzyme activity.
Parameters Artificial Milk Replacers (AMR) SEM p-Value
RMR KMR MMR
Internal organs characteristics (g/live body weight)
Liver 4.46 4.00 3.81 0.162 0.25
Spleen 0.14 0.10 0.10 0.015 0.51
Kidney 1.24 1.18 1.12 0.046 0.61
Thoracic organs 11.07 1.09 1.15 0.063 0.38
Pancreas 0.06 0.07 0.05 0.007 0.99
Full stomach 7.40 7.44 7.22 0.555 0.79
Stomach wall 2.09 2.01 1.99 0.061 0.65
Intestinal organs 27.38 6.87 7.37 0.242 0.54
Full caecum 16.3 16.1 16.2 0.422 0.97
Caecal wall 2.75 2.49 2.34 0.134 0.48
Caecal pH 6.50 6.30 6.38 0.081 0.63
Caecal digestive enzyme activities (U)
Amylase 12.2 ab 18.5 b10.4 a1.41 0.04
Protease (×10−1) 7.26 b6.84 ab 5.79 a0.253 0.04
Cellulase 3.71 3.50 3.60 0.057 0.31
Duodenal villi morphology (µm)
Villus height 320 a361 ab 380 b8.50 0.04
Villus crypt 66.2 ab 62.6 a68.4 b0.737 0.007
RMR =Rabbit milk replacer, which was used in this study, KMR =Kitten milk replacer (KMR
®
, Pet-Ag Inc.,
Hampshire, IL, USA), MMR =Mammal milk replacer (Zoologic
®
Milk matrix 30/52, Pet-Ag Inc., Hampshire, IL,
USA), SEM =pooled standard error of mean, BW =Body weight, FI =Feed intake, ADG =Average daily weight
gain, FCR =Feed conversion ratio;
a, b
The differences in superscript letter in the same row represented statistical
significant differences (p<0.05);
1
Thoracic organs includes lungs and heart;
2
Intestinal organs includes stomach,
small intestine, large intestine, caecum and rectum with content.
The average number of faeces pellets from two rabbits of each replicate in the experiments is
illustrated in Figure 1and Table A1. The graph shows a steady increase in the number of faecal pellets
with increasing age (p<0.001); however, a sharp drop occurred in all study groups at 35 days of age,
followed by another increase. The largest number of faecal pellets existed in the MMR group compared
to the RMR group (p<0.05; Appendix ATable A1), with the KMR group between them (p>0.05).
A significant interaction between the fixed factors (age and treatment group) was identified (p<0.05).
Generally, the same increasing trend was observed in all groups, except for the sharp rise in the number
of faecal pellets in the MMR, RMR and KMR groups at 22–23, 24–26 and 34–36 days of age, respectively.
Animals 2020, 10, x 7 of 12
Figure 1. Effect of different milk replacers on number of faecal pellets (RMR, rabbit milk replacer in
this study; KMR, kitten milk replacer, KMR®, Pet-Ag Inc., Hampshire, IL, USA; and MMR, mammal
milk replacer, Zoologic® Milk matrix 30/52, Pet-Ag Inc., Hampshire, IL, USA).
4. Discussion
The RMR was formulated according to the profile of rabbit milk; therefore, its chemical
composition was the most similar to the composition of rabbit milk [10]. KMR contained a higher
proportion of crude protein and was lower in fat and energy than RMR and rabbit milk. On the other
hand, MMR was lower in crude protein and higher in fat and energy than RMR and rabbit milk. A
high density of nutrients and energy was the unique characteristic of rabbit milk, which contained
respectively around four and three times higher proportions of protein and lipids than cow’s milk
[10]. A short milking time is a common nursing behaviour, explaining the high density of rabbit milk
[6]. The amount of milk replacer fed to young rabbits was calculated on the basis of stomach capacity.
Milk replacer in powder form was used for all formulations in this study because a highly
concentrated milk mixture can be formulated from dried powder but not in liquid form [8,9]. Rabbit
milk protein comprises around 70% and 30% casein and whey protein, respectively [10]. Therefore,
casein served as a major protein ingredient in the milk replacer formulation for RMR and the two
other commercial milk replacers, whereas dried skimmed milk powder represented another protein
source, which was used in a lower proportion than casein. Respectively, either whey or milk protein
concentrate was supplemented in KMR and MMR, whereas in RMR they were not. A low level of
lactose is present in rabbit milk; therefore, cow’s or goat’s milk is limited as a milk replacer
formulation [10]. Moreover, lactase activity in rabbits decreases with age and does not respond to the
lactose concentration in the diet. Therefore, excessive lactose intake can lead to a digestive disorder
[6]. Differences in the type of raw protein source and quantity influence the diversity of the amino
acid profile. Although the amino acid profile was not evaluated in this study, the intake of amino
acids would have been sufficient for rabbits because casein, considered an ideal protein, was used as
the main ingredient and the percentage of crude protein in all milk replacers was higher than nutrient
requirements [4,6].
Fat in rabbit milk represents the major energy sources for rabbits [10], whereas excessive starch
intake promotes digestive problems and increases the mortality rate of young rabbits [4]. The highest
nitrogen-free extract was present in the KMR formulation; however, no adverse effects were observed
in this group. Medium-chain fatty acids, mainly caprylic (C8:0) and capric acid (C10:0), were the
major components of fatty acids in rabbit milk, comprising around 50% of total fatty acids [10].
Vegetable oil or hydrogenated palm oil served as the only lipid sources in milk replacers, comprising
a high proportion of polyunsaturated fatty acids; however, they still contained these medium-chain
fatty acids. Another function of caprylic and capric acid was their antibacterial properties, which
maintained microbial community development and prevented pathogen invasion. Supplementation
Figure 1.
Effect of different milk replacers on number of faecal pellets (RMR, rabbit milk replacer in
this study; KMR, kitten milk replacer, KMR
®
, Pet-Ag Inc., Hampshire, IL, USA; and MMR, mammal
milk replacer, Zoologic®Milk matrix 30/52, Pet-Ag Inc., Hampshire, IL, USA).
Animals 2020,10, 1087 7 of 12
4. Discussion
The RMR was formulated according to the profile of rabbit milk; therefore, its chemical composition
was the most similar to the composition of rabbit milk [
10
]. KMR contained a higher proportion of
crude protein and was lower in fat and energy than RMR and rabbit milk. On the other hand, MMR
was lower in crude protein and higher in fat and energy than RMR and rabbit milk. A high density of
nutrients and energy was the unique characteristic of rabbit milk, which contained respectively around
four and three times higher proportions of protein and lipids than cow’s milk [
10
]. A short milking
time is a common nursing behaviour, explaining the high density of rabbit milk [
6
]. The amount of milk
replacer fed to young rabbits was calculated on the basis of stomach capacity. Milk replacer in powder
form was used for all formulations in this study because a highly concentrated milk mixture can be
formulated from dried powder but not in liquid form [
8
,
9
]. Rabbit milk protein comprises around
70% and 30% casein and whey protein, respectively [
10
]. Therefore, casein served as a major protein
ingredient in the milk replacer formulation for RMR and the two other commercial milk replacers,
whereas dried skimmed milk powder represented another protein source, which was used in a lower
proportion than casein. Respectively, either whey or milk protein concentrate was supplemented
in KMR and MMR, whereas in RMR they were not. A low level of lactose is present in rabbit milk;
therefore, cow’s or goat’s milk is limited as a milk replacer formulation [
10
]. Moreover, lactase activity
in rabbits decreases with age and does not respond to the lactose concentration in the diet. Therefore,
excessive lactose intake can lead to a digestive disorder [
6
]. Differences in the type of raw protein
source and quantity influence the diversity of the amino acid profile. Although the amino acid profile
was not evaluated in this study, the intake of amino acids would have been sufficient for rabbits
because casein, considered an ideal protein, was used as the main ingredient and the percentage of
crude protein in all milk replacers was higher than nutrient requirements [4,6].
Fat in rabbit milk represents the major energy sources for rabbits [
10
], whereas excessive starch
intake promotes digestive problems and increases the mortality rate of young rabbits [
4
]. The highest
nitrogen-free extract was present in the KMR formulation; however, no adverse effects were observed
in this group. Medium-chain fatty acids, mainly caprylic (C8:0) and capric acid (C10:0), were the major
components of fatty acids in rabbit milk, comprising around 50% of total fatty acids [
10
]. Vegetable
oil or hydrogenated palm oil served as the only lipid sources in milk replacers, comprising a high
proportion of polyunsaturated fatty acids; however, they still contained these medium-chain fatty
acids. Another function of caprylic and capric acid was their antibacterial properties, which maintained
microbial community development and prevented pathogen invasion. Supplementation with these
medium-chain fatty acids should confer health benefits on rabbits. Nevertheless, no health problems
occurred in this study, although these medium-chain fatty acids were not supplied in the formulations.
The essential fatty acids were supplied in sufficient amounts to fulfil the nutritional requirements of
all the study groups, as there was a very high amount of fat in all the formulations [
6
]. However,
an antioxidant such as tocopherol should be supplemented to prevent lipid oxidation. The minerals
and vitamins in RMR were higher than the minimum requirements in rabbits [
6
]. In the early
period of the experiment, the rabbits received nutrients and energy from the milk replacers in high
proportions compared to the pelleted diet, which represented only around 1/3 of the total intake in
the first period. Subsequently, a higher intake of the pelleted diet became the main nutrient and
energy source. Growth performances did not differ between treatment groups, and no morbidity or
mortality of rabbits occurred during or after the experimental period. Moreover, the rabbits’ final
body weights were similar to those of their counterparts reared with their mother at the same age,
as reported in the study of [
5
]. These findings demonstrate the potential of using milk replacers in
early weaned rabbits. However, the low energy and nutrient requirements of the rabbits in this study
were characteristics of a native breed with a slow growth rate compared to hybrid or commercial meat
rabbits [
5
]. Therefore, milk replacers can certainly be used in local breed or pet rabbits, possibly offering
benefits to veterinarian and pet rabbit producers. Rabbits raised in intensive rearing systems and/or
with high growth rates may be studied further as to which milk replacer can support their growth
Animals 2020,10, 1087 8 of 12
performances and the maintenance of a healthy condition. Another study [
9
] reported lower growth
performances in commercial meat rabbits fed KMR compared with rabbits fed rabbit milk.
A previous report has shown that the gut microbial community of rabbits plays an important role
relating to the efficiency of nutrient fermentation, productive performance and heath condition [
19
].
A sterile gut is observed in rabbits immediately after birth. Subsequently, microbes slowly and
continuously colonize the rabbit gut and the amount of antimicrobial substances in rabbit milk and
nutrients have major effects on the development of gut functions and the microbial community [
10
,
19
].
A large variation in the bacterial community was observed between young rabbits at an early age.
However, the composition of the bacterial community between rabbits became more similar with
increasing age, especially with pelleted diets [
13
,
19
]. To our knowledge, the caecum is a major
organ of bacterial fermentation in rabbits, providing around 50–60% of daily energy requirements.
The caecal environment greatly affects the microbial community in rabbits, especially in terms of
pH, being influenced in a major way by fermentable products called volatile fatty acids. A low
nutrient fermentation efficiency can result in a higher value of caecal pH, increasing the risk of a
digestive disorder [
6
]. The caecal pH of the RMR group seemed to be higher than that of the other
groups. However, the caecal pH of the RMR group was lower than 6.73, insufficient to promote
a gut health problem [
6
]. Moreover, there was no statistically significant difference in caecal pH,
growth performance, digestibility, morbidity and mortality during the experiments, supporting the
possibility of using RMR in rabbits.
Caecal enzyme activity was another indicator representing microbial activity. The amount of caecal
amylase and protease in the RMR and KMR groups was higher than in the MMR group. Fermentation
products from probiotic bacteria and prebiotics play an important role in supporting the development
of a normal flora in rabbits [
20
]. Supplementation with fermentation products from several normal
flora bacteria (Lactobacillus sp., Enterococcus faecium,Bifidobacterium bifidum,Pediococcus adicilactici)
and prebiotics (fructooligosaccharide) in the KMR formulation contributed to higher enzyme activity,
whereas RMR and MMR did not contain these supplements. Such findings are in accordance with
another research study [
8
]. A higher survival rate was observed in the milk replacer with probiotics
and prebiotics (35.3%) compared to without these additions (21.3%) in infant cottontail rabbits in which
the chemical composition of these milk replacers were similar [
8
]. Thus, probiotics and/or prebiotics
may have been the key factor determining this result. The unsuitable chemical components of KMR
with respect to rabbit milk and the slow growth rate of bacteria in the first period could have been
the cause of the lower feed intake in the first period in the KMR group. Subsequently, feed intake
in the KMR group was higher than in the others at the end of the experiment, as a consequence of
the full development of microbes. The closeness of the chemical component of RMR to that of rabbit
milk may have promoted the appropriate substrate for early microbial colonization, leading to higher
caecal enzyme activity. None of prebiotic supplement and difference in chemical composition in MMR
could be the cause of the lower enzyme activity in the caecum compared to other groups. Therefore,
supplementation with probiotics and/or substrates for a normal flora in feed formulation with the
appropriate chemical components (i.e., in line with rabbit milk) may represent the best procedure to
achieve good development of the microbial community in early weaned rabbits. However, a microbial
community analysis should be performed in a future study to confirm this hypothesis.
No statistically significant difference in growth performance was observed in this study. However,
a trend existed that can be explained in detail. The lowest FCR was observed in the RMR group as
a consequence of the lower ADFI and the higher ADG. This may have been due to the appropriate
crude protein and fat proportion in the RMR, which promoted lower adaptation following the milk
replacer in the first period. However, the highest ADFI was observed in the KMR group as the
lowest energy density because the feed intake was stimulated by a physiological function until the
animal had obtained its daily energy requirement [
6
]. Therefore, a lower pelleted diet intake could be
observed in the group already receiving a high-density milk replacer in the RMR and MMR groups.
The digestibility of dry matter and nutrients in the RMR group tended to be lower than in the other
Animals 2020,10, 1087 9 of 12
groups. The supplementation of prebiotics and substrates for microflora in KMR can support the
microbial community and facilitate increased digestibility. In addition, MMR contained the highest
energy density, which bacteria can use as energy sources to produce butyrate. Intestinal epithelial cells
can utilize such short-chain fatty acids as an energy source, promoting proliferation, differentiation and
gut immunity [
21
]. Thereby, the greatest villus height and villus crypt depth were seen in the MMR
group, enabling better digestion and absorption and providing higher nutrient digestibility RMR as a
result [
22
]. Furthermore, the amount and characteristics of hard faeces can be used to indicate digestive
health [
7
]. The total weight of faecal excretion did not differ between groups, but the largest number of
faecal pellets was found in MMR, followed by KMR and RMR. Moreover, no soft faeces were found
under the rabbit cage, indicating that crude protein intake did not exceed their requirement [19].
Lower growth performance and survival rates were reported in the group fed with KMR compared
with the group fed with rabbit milk from lactating does [
8
]. Unfortunately, this study did not compare
the milk replacers with rabbit milk. Early separation at 14 days of age may have been the cause of the
negative consequences found in the study of [
8
], whereas separation at 18 days of age did not result in
any serious adverse outcomes here. In addition, a high mortality rate was observed in the study of [
8
]
because the rabbits were too young, were injured and were experiencing high levels of stress due to
being wild rabbits.
5. Conclusions
Differences in efficiency between the RMR developed in this study and commercial milk replacers
(KMR and MMR) used in 18-day-old rabbits were revealed in the current study. Based on the
results, it was possible to use RMR as a milk replacer for 18-day-old rabbits and to wean at 36 days
of age, this not providing any adverse consequences for final body weight, ADG, FCR, ADFI,
nutrient digestibility, internal organ characteristics, caecal pH, amount of faeces excretion and crude
protein intake. Lower nutrient digestibility was observed in the RMR group without statistically
significant differences. Therefore, probiotics and/or prebiotics can be supplemented in formulations to
promote a suitable microbial community and to provide benefits in terms of growth performance and
nutrient digestibility.
Author Contributions:
Conceptualization, A.K.; methodology, A.K.; investigation, P.C. (Panthiphaporn
Chankuang), A.L., K.J., C.K., T.S., P.C. (Pipatpong Chundang) and A.K.; resources, P.S. and C.Y.; data curation, A.K.;
writing—original draft preparation, A.K.; writing—review and editing, P.C. (Panthiphaporn Chankuang), A.L.,
K.J., C.K., T.S., P.C. (Pipatpong Chundang), A.K., P.S. and C.Y.; project administration, A.K.; funding acquisition,
A.K. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by Student Development Fund, Faculty of Veterinary Medicine, Kasetsart
University, Bangkok, Thailand.
Acknowledgments:
The authors would like to thank Faculty of Veterinary Medicine, Veterinary Teaching Hospital
Kamphaengsaen campus (Kasetsart University, Nakhon Pathom, Thailand) and Mr.Saha Bairak (Saha farm,
Kanchanaburi, Thailand) for providing the rabbit milk replacer and rabbits, respectively. Moreover, we would like
to acknowledge Thanaporn Sriprathardtrakul, Jiraphat Wangka and Sirapoom Narktap for the support in rabbit
rearing and feeding.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A
Table A1. Effect of different milk replacers on number of faecal pellets.
Artificial Milk Replacers Age (days) Number of Feces
RMR A
19 0 a
20 0 a
21 6ab
22 117 b
Animals 2020,10, 1087 10 of 12
Table A1. Cont.
Artificial Milk Replacers Age (days) Number of Feces
23 244 cd
24 263 c
25 338 def
26 401 efgh
27 379 defg
28 385 defg
29 371 cde
30 543 hi
31 548 fgh
32 590 hi
33 592 ghi
34 454 def
35 587 ghi
36 742 i
KMR AB
19 75.6
20 70.8
21 191
22 180
23 418
24 432
25 435
26 456
27 421
28 428
29 378
30 493
31 467
32 505
33 441
34 431
35 519
36 563
MMR B
19 0
20 3.33
21 52.3
22 151
23 311
24 245
25 426
26 509
27 444
28 432
29 406
30 520
31 463
32 476
33 497
34 350
35 436
36 477
SEM 12.02
p-Value
Groups 0.03
Age 0.001
Groups ∗Age 0.02
RMR =Rabbit milk replacer in this study, KMR =Kitten milk replacer (KMR
®
; Pet-Ag Inc., Hampshire, IL, USA),
MMR =Mammal milk replacer (Zoologic
®
Milk matrix 30/52; Pet-Ag Inc., Hampshire, IL, USA; SEM =Pooled
standard error of mean;
a–i, A, B
The differences in superscript capital or lower case letter in the same column represent
statistical significant differences of groups or age, respectively (p<0.05).
Animals 2020,10, 1087 11 of 12
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