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Unveiling the attributes of rabbit milk

Article  in  animal · May 2023

DOI: 10.1016/j.animal.2023.100848

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Unveiling the attributes of rabbit milk

A. Ludwiczak, J. Składanowska-Baryza, B. Kuczyńska, E. Sell-Kubiak, M.

Stanisz, E. Skrzypczak

PII: S1751-7311(23)00144-1
DOI: https://doi.org/10.1016/j.animal.2023.100848
Reference: ANIMAL 100848

To appear in: Animal

Received Date: 3 January 2023

Revised Date: 30 April 2023
Accepted Date: 2 May 2023

Please cite this article as: A. Ludwiczak, J. Składanowska-Baryza, B. Kuczyńska, E. Sell-Kubiak, M. Stanisz, E.
Skrzypczak, Unveiling the attributes of rabbit milk, Animal (2023), doi: https://doi.org/10.1016/j.animal.
2023.100848

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Title

Unveiling the attributes of rabbit milk

A. Ludwiczaka, J. Składanowska-Baryzaa, B. Kuczyńskac, E. Sell-Kubiakb, M. Stanisza,

E. Skrzypczaka
aDepartment of Animal Breeding and Product Quality Assessment, Poznań University
of Life Sciences, Słoneczna 1, 62-002 Suchy Las, Poland
bDepartment of Genetics and Animal Breeding, Poznań University of Life Sciences,
Wołyńska 33, 60-637 Poznań, Poland
cAnimal Breeding Department, Faculty of Animal Breeding, Bioengineering and
Conservation, Warsaw University of Life Sciences, Ciszewskiego 8, 02-786 Warsaw,
Poland

Corresponding author: Agnieszka Ludwiczak. Email:

agnieszka.ludwiczak@up.poznan.pl

Abstract

Increasing the knowledge of rabbit milk can help in breeding practice to solve issues
considering the health and growth of rabbit kits. The goal of the study was to perform
a broad physicochemical analysis of rabbit milk and examine the effect of the
reproductive status of the females on daily milk yield and milk attributes. The study was
conducted on a commercial rabbit farm and included three consecutive lactations of
Hycole does. It has been observed that the daily milk production increased from the
2nd till the 14th day of lactation when does produced almost 300 g of milk daily. The
day of lactation caused a significant variation in the content of total solids, solids-not-
fat, total protein, casein, lactose, C18: 2, C18: 3, Somatic Cell Count, and pH. The
percentage of fat globules categorized according to their diameter changed with the
ongoing lactation as well, and the diameter increased from 5 to 7 µm. The percentage
of small milk fat globules decreased with lactation day, causing a possible decrease in
the digestions rates of milk. Pregnancy had a negative impact on milk production, kits
growth performance, and the content of total protein, solids-not-fat, and lactose in milk.
Therefore, we can speculate about the negative impact of overlapping lactations and
pregnancies on rabbit kits, as their growth is dependent on milk production and
composition.

Keywords: reproductive status; pregnancy; milk yield; fat globules; fatty acids
Implications

The physicochemical attributes of rabbit milk are affected by multiple intrinsic and
extrinsic factors and have not been examined to a sufficient degree. Thus, the aim of
this study is to conduct a broad examination of rabbit milk in the scope of its
physicochemical traits (basic chemical composition, SCC, pH, fat globules
characteristics) and nutritional quality (fatty acid profile). We also examined the effect
of reproductive status on milk quality and the daily milk yield of the doe. We showed
that most of the attributes of rabbit milk change with the course of lactation, with some
of them affected by the consequent pregnancy. Moreover, we observe a negative
impact of late pregnancy on milk production and on the kits’ growth performance in the
nursing period. Thought the milk physicochemical traits were affected by the
reproductive status of does to a limited extent.

Introduction

The nursing behavior and the physiology of milk production in rabbits are unique and
rarely appreciated by rabbit producers. Among the facts that we know about lactation
in farmed rabbits the following can be named: 1. a female rabbit is able to produce
around 7 kg of milk over a 28-day lactation period; 2. the rearing success of rabbits
depends on the amount of produced milk and its basic chemical composition, and 3.
the content of essential fatty acids in milk affects the growth and survivability of young
in the nursing period (Maertens et al., 2006a,b). The topics not examined in farmed
rabbits are milk fat globules in rabbit milk and the relation between the reproductive
state of the female and the daily milk yield and milk quality (including fatty acids profile).

As for the milk fat globules (MFG), the mammary glands secrete over 95% of lipids in
this form (Månsson. 2008; Chai et al., 2022). This may be an important fact from the
perspective of rabbit kits’ survival and growth in the early post-partum period, since the
physical structure, size, and granulometry of MFG are known to affect their digestion
(Faustini et al., 2010; Baumgartner et al., 2017). As for the physicochemical traits of
milk, including the chemical composition, we may assume that in rabbits, just like other
mammals, milk is affected by the nutrition and physiology of the animal (Penasa et al.,
2016; Suarez-Trujillo et al.. 2021), though the fatty acid (FA) composition in milk is
mainly affected by the diet (Jin et al., 2017; Więcek et al., 2018), and to some extent
by genetic predispositions (Kęsek et al., 2014; Conte et al., 2010).
Unlike in other mammalian species, in farmed rabbits the relations between the
lactation day, milk yield, and milk chemical composition were studied to a limited
extent, motivating us to examine this topic. Moreover, the effect of pregnancy on milk
production and quality has not been studied in rabbits, though knowing these relations
could be important in understanding how the overlapping lactations and pregnancies
contribute to energy deficit development and kits growth and mortality.

Increasing the knowledge of rabbit milk composition can help in breeding practice to
solve issues considering the health and growth of rabbit kits in the pre and post-
weaning period. Thus, the aim of this study is to conduct a broad examination of rabbit
milk in the scope of its physicochemical traits (basic chemical composition, SCC, pH,
fat globules characteristics) and nutritional quality (fatty acid profile). We also
hypothesized about the possible effect of the reproductive status of the doe on the milk
yield and milk attributes.

Material and methods

Animals and housing conditions

Permission from the Local Ethic Commission was not required for this study according
to the Journal of Laws of the Republic of Poland, Item 266, Article 1 of the Act of 15
January 2015 on the Protection of Animals Used for Scientific and Educational
Purposes in Poland. The proper statement of the Local Ethic Commission is available
upon request.

The study was conducted on a commercial rabbit farm, and the animals’ housing
conditions were similar to those previously described by Ludwiczak et al. (2020). The
microclimate on the farm was controlled (temperature 16–20 ᵒC, humidity 60-75%,
lighting schedule of 12h light/12h darkness, or 16h light/8h darkness 5 days before the
insemination). The trial was performed on 120 Hycole does in the same reproductive
cycle, managed as one batch. Their first artificial insemination (AI) was conducted at
the age of 17–19 weeks and was repeated every 42 days, with the pregnancy
confirmed between 10 to 12 days after the AI. The study covered the period starting
before their 3rd kindling and ending after the 5th kindling. The number of successfully
inseminated and discarded does is presented in Table 1. Rabbit does and their kits
were kept in individual wire-mesh cages including a feeder, nipple drinker, and a
closable nest box. The cage dimensions were 40 cm x 85 cm x 35 cm (width x length
x height). Each cage was connected to a nest (30 cm x 40 cm x 30 cm). Throughout
the examined period, the rabbit does were fed ad libitum with a commercial pelleted
feed composed of soybean meal, wheat bran, alfalfa, beet pulp, wheat, barley,
soybean oil, sodium chloride, calcium carbonate, monocalcium phosphate (Table 2).
They received a diet for lactating females from day 21 of pregnancy till day 26 of
lactation. From day 26 of lactation, this feed was mixed with feed for kits in the pre and
post-weaning period, and replaced by it from day 28 of lactation. The females and their
kits had unlimited access to fresh water. During the examined period (3rd-5th parity),
semen of the white male line (Mâle Blanc HYCOLE; white or white with black
extremities; adult weight: 6.5-7.5 kg) was used for AI. After birth, litters were
standardized by cross-fostering to 9 kits per nest. From the moment of standardization
until insemination the nests were opened only once daily. From day 15th of lactation,
the kits had unlimited access to the mother, drinker, and feeder. For the purpose of
this research, one exception was made. In order to measure the daily milk production
and collect milk samples, the kits were separated from their mothers from 20:00 on
day 20 till 9:00 on day 21 of lactation. Throughout this time the kits had unlimited
access to the drinker and feeder. The kits were weaned at the age of 35 days, by
removing the mother from the cage.

Reproductive performance and milk examination

Reproductive traits examination and milk recovery

The traits describing the females and their kits i.e. body weights, litter size, and
performance, and daily milk yield were collected to have a full overview of the research
material. The daily milk yield on days 2, 9, and 14 of lactation was measured with the
method developed by Lebas (1968), by recording the body weight of the does before
and after nursing and calculating the difference. Rabbit milk recovery and chemical
composition examination were conducted using the methodology described in the
study of Ludwiczak et al. (2020). The samples were collected during each of the 3
examined parities, on the 2, 9, 14, and 21 days post-partum, and the does used for
milk recovery were not included in the daily milk production evaluation. A quantity of
ten ml of milk per rabbit doe was obtained into a plastic test tube by gently massaging
the mammary glands, and chilled to 4 ᵒC directly after recovery. Samples were
transported under chilled conditions to the laboratory for examination of pH, MFG,
SCC, basic chemical composition (total solids, solids-not-fat, fat, protein, casein,
lactose, and ash), and fatty acids profile. During each milk recovery, the milk samples
were collected from 30 randomly selected does from the examined parity (the reason
for that low number of females was the fact that the procedure requires time, and the
time was limited because it was a commercial farm). On day 21 of lactation, the milk
was collected after pregnancy confirmation, from both, pregnant and non-pregnant
does. If the number of non-pregnant does was less than 15, then the pregnant group
selected for milk recovery was bigger than the non-pregnant (we wanted to keep the
total sample size of 30 milk samples per lactation day).

Milk physicochemical traits examination

The pH was measured by immersing an InLab® Power electrode (Mettler Toledo,

Urdorf, Switzerland) combined with a mobile pH‐meter (Mettler Toledo, Urdorf,
Switzerland, type 1140) and a temperature probe (Pt 1000, Urdorf, Switzerland). For
calibration of the pH equipment, buffers of pH 4.0 and 7.0 at room temperature were
used. The somatic cell count in colostrum and milk was performed using the flow
cytometry method by means of the Bentley Somacount 150 instrument (Bentley,
Warsaw, Poland). The chemical composition of milk was determined by automated
infrared analysis with a MilkoScan FT 120 analyzer (Foss Electric, Warsaw, Poland).
The MilkoScan FT 120 analyzer was calibrated immediately before the analysis using
internal test procedures and samples standardized for the content of total protein and
total fat in accordance with the reference methods of Kiejdal and Röse-Gotlieb in the
test laboratory of the Department of Animal Breeding of the Warsaw University of Life
Sciences. Fatty acid content in rabbit milk as assessed by direct methylation described
by Trigueros and Sendra (2015). Individual fatty acids were identified in crude fat using
an Agilent 7890A GC (Agilent, Waldbronn, Germany) according to Kuczyńska et al.
(2012). Each peak was identified using pure methyl ester standards: FAME Mix RM–
6, Lot LB 68242; Supelco 37 Comp. FAME Mix, Lot LB 68887; Methyl linoleate, Lot
094K1497 (Supelco, Bellefonte, PA, USA). The measurements of fat globules in rabbit
milk were performed according to the method developed by Martini et al. (2013), by
means of a microscope (Motic, BA410E) equipped with a digital camera. The diameter
(μm), and the number of fat globules per ml of milk were measured by means of the
Motic Images Plus 3.0 software. An amount of 5 μl of milk was placed on a microscope
slide and mixed with 3 μl of Sudan III. The photos of fat globules were made at 1000x
magnification, and the measurements were performed in two fields of vision. The
globules were grouped according to their diameter: small (<4 μm), medium globules
(4≥ and ≤9 μm), and large (>9 μm). The amount of each of the globules category is
given as a percentage of the total number of globules counted by means of a Thoma
cell counting chamber. The chamber is divided into 16 big squares, with 16 small
squares in each of the big ones. The globules were counted in 4 small squares of the
chambers, and the mean values were used to calculate the number of globules in 1
cm3 of milk, according to the formula:

L= a*4*106

where:

a – mean number of globules in one small square of the Thoma chamber

4*106 = K – a constant value of the chamber

Statistical analysis

All statistical analyses were made with Statistica ver 14.0.0.15 (Statsoft) tools. The
fertility rate was calculated as the percentage of females with pregnancy confirmed
through palpation compared to the total number of does submitted to AI. The kindling
rate was calculated as the percentage of does that gave birth compared to the number
of inseminated does. The collected data were checked for normality and variance
uniformity. Initially, the general linear models were used to calculate the effect of parity
order, lactation day, and parity order * lactation day interaction on the reproductive
traits, kits growth, and milk quality traits. The observations were repeated for a batch,
not individually. No effect of parity and no interaction was found, therefore, in order to
simplify the presentation of the results, the model was changed, and the one-way
ANOVA was used with the effect of lactation day only on the examined traits. The effect
of reproductive status (pregnant vs. non-pregnant females) on selected traits
measured on day 21 of lactation was examined with one-way ANOVA. The statistical
model is presented as Supplementary Material S1. Moreover, Pearson correlations
between litter size and growth performance for pregnant and not pregnant does was
estimated.

Results

Reproductive traits and growth of kits

On the basis of the palpation and kindling results, a fertility rate of 85.6%, and a kindling
rate of 83.9% were observed. The characteristic of the examined does and their litters
are presented in Table 3. The lowest body weight of does was noted on day 2 after
kindling, then it reached a peak on day 9 of lactation, and decreased again between
day 9 and 14 of lactation. The number of kits per litter (referred to as litter size)
significantly decreased between day 2 and day 14 (9.0 vs 8.2; P=0.004), whereas it
remained constant on days 14 and 21. The daily milk production increased by 52.7%
between day 2 and 9 of lactation, and by 16.1% between 9 and 14 days. Around 14th
day does reached the lactation peak, producing almost 300 g of milk daily. Between
14 and 21 days of lactation, the daily milk production decreased, reaching 205.2 g on
the 21st day of lactation. The pregnancy did not affect the female body weight or litter
size measured on day 21st of lactation. Non-pregnant females were characterized by
significantly greater milk production (221.8 vs 180.2 g; P=0.008) and litter body weight
on day 21 (3226 vs 3012 g; P=0.042) compared to pregnant does and by greater
growth performance of kits on days 2 to 21 (Table 4).

We have also estimated the Pearson correlation coefficients between the litter size on
days 2 and 21, litter weight, and litter weight gain (Table 5). In both groups of females
(pregnant and not pregnant), highly significant correlations were found between litter
size on day 2 and litter weight on day 21 (0.931; P=0.0001), and litter size on day 21
with litter weight gain 2-21 days. In pregnant females, litter weight on day 2 was
significantly correlated with litter weight gain 2-21 but not as highly correlated as in
non-pregnant females (0.699 vs 0.991).

Rabbit milk attributes

Chemical composition

Table 6 contains the basic physicochemical characteristics of rabbit milk. The SCC
increased with lactation day and was almost twice as higher on days 14 and 21 (168.4
and 173.9^103 mL-1) compared to day 2 post-partum (97.44^103 mL-1). From the
analyzed chemical compounds, only the content of total solids and fat was not affected
by the lactation day. Total protein was significantly higher on day 9 (10.74%) compared
to day 2 (10.01%; P=0.017). SNF, lactose, and casein content were the greatest on
day 9. The pH also changed during lactation. The pH of milk collected on day 2 post-
partum was significantly greater compared to milk from days 14 and 21 (7.01 vs 6.82
and 6.80; P=0.000). According to the data presented in Table 7, pregnancy affected a
limited number of traits. Milk from pregnant does had a higher content of total protein
(11.05 vs 10.12%; P=0.008), SFN (16.12 vs 14.01%; P=0.002), and lactose (2.09 vs
1.87%; P=0.003) compared to not pregnant females.

Milk fat globules

In order to compare MFG from rabbits with literature data of other mammalian species,
the mean diameter independent of the lactation day and reproductive status of the doe
was calculated and was 5.88 (±0.10) µm. MFG had a greater diameter on days 14 and
21 compared to the other two days of lactation. The number of small globules was
significantly greater on day 2 compared to day 14 (by 14.8 percentage points). It was
just the opposite in the case of the medium and big globules, as their percentage
increased with the lactation day, and was much greater on day 14 compared to day 2.
The reproductive status of does had no effect on the MFG characteristics (Table 7).

Fatty acids profile

The MUFA constitutes almost 13% of total FA in rabbit milk, while PUFA is over 9%
and SFA is over 75% (Table 8). Among the PUFA, the greatest content reaching over
9% was noted for the linoleic acid. MUFA were represented mainly by the oleic acids,
while among SFA, the caprylic, capric, and palmitic acids were in the greatest amount.
The lactation day showed a limited effect on the fatty acid composition of rabbit milk.
The milk was characterized by the highest content of C18:2 on day 2 of lactation (9.76
nmol/100g; P=0.047), and a decreasing content of C18:3 from day 2 to day 21 of
lactation (from 2.76 to 2.10 nmol/100g; P=0.032). As a result, the PUFA content was
influenced by the lactation day and decreased from day 2 to day 14. We have not
presented the influence of pregnancy on the fatty acid profile of rabbit milk, as no such
effect was found.

Discussion

The goal of this study was to thoroughly examine the physicochemical traits (SCC,
basic chemical compounds content, fatty acid profile, and fat globules diameter and
percentage according to the size) of milk from rabbits housed under intensive
conditions, and evaluate the variation of these traits according to the lactation day and
effectiveness of the AI. There are three main innovative aspects of the presented study:
1. We gathered data on the attributes of rabbit milk using the most recent available
technology of milk analysis; 2. We have examined how all the aforementioned
attributes change with lactation day; 3. We have checked the influence of the doe’s
reproductive status on daily milk production and milk attributes.

In this study, we used the term milk, not colostrum from the 2nd day of parturition
because in rabbits the colostrum is produced only a few hours after kindling. We have
reported the colostrum composition in our previous studies (Ludwiczak et al., 2020 and
2021), and it strongly differs from milk collected on day 2 of lactation. The chemical
composition of rabbit milk is difficult to compare with the literature, as the lactation days
of milk acquisition differ among studies and species, as well as the methods of rabbit
milk analysis. We have noted a similar range of main chemical component content
between the presented study and our previous work (Ludwiczak et al., 2020 and 2021).

In the review of Maertens et al. (2006), the mean values of dry matter, protein, fat, and
lactose content for the first 3 weeks of lactation are 29.8 g/100g, 12.3 g/100g, 12.9
g/100g, and 1.7 g/100g. However, it should be noted that the values were calculated
as mean on the basis of only three different research papers. As for the milk
composition of other Glires, the rat’s milk has a greater content of total fat (12.8–15.8
g/100ml; Mozeš et al., 1993) and lower level of protein (8.9–9.2 g/100 ml; Mozeš et al.,
1993) compared to rabbit milk analyzed in this study. We have observed changes in
rabbit milk’s basic composition with the day of lactation. Generally, the content of most
of the components (TS, SNF, TP, casein, and lactose) increased from 2 to 9 days of
lactation, decreased from day 9 to day 14, and remained constant between days 14
and 21. Simultaneously, daily milk production increased significantly from day 2 to day
14 and decreased from day 14 to day 21. This observation was in accordance with
other research results in the field of rabbit milk (Kolawole et al., 2013; Ludwiczak et al.,
2020). The SCC values measured in our study increased with the day of lactation. This
phenomenon is also observed in milk cows and related to exposure of mammary
glands to pathogens, glandular damage, and the dilution effect as well (Hagnestam-
Nielsen et al., 2009; Craig et al., 2022). In other Glires, like rats, the changes in milk
chemical composition with ongoing lactation were also observed, but without a clear
pattern (Mozeš et al., 1993).

Although the fatty acid profile of rabbit milk has been reported in a number of studies
(Maertens, et al. 2006b), no studies on the effect of lactation day on the fatty acids
profile in rabbit milk are available. In other mammalian species, like cows, the lactation
phase affects the content of fatty acids in milk, in particular C4:0 and C12:0 at the onset
of lactation (Auldist et al., 1998). It can be explained by physiological mechanisms, as
the onset of lactation (and the negative energy balance) triggers adipose tissue
mobilization. We have also noted an effect of lactation day on some of the detected
FA, including C18:2, and C18:3. According to Betancourt López, et al. (2019) the
caecotroph and rabbit milk FA profiles are significantly correlated, especially for 12:0,
14:0, 15:0, 16:0, 18:0, 18:1 trans-6/8, 18:1 trans-9, 18:1 cis-9, 18:2 n-6, 18:3 n-3, 20:1
cis-9, 20:2 n-6, and 22:0. The authors have proved evidence of cecum
biohydrogenation processes, as the content of saturated FA found in the caecotrophs
was greater compared to feces.

Maertens et al. (2006b) showed that the rabbit milk fatty acid profile is characterized
by a high level of caprylic acid (C8:0; 26.3% of total FA) and capric acid (C10:0; 20.1%
of total FA), and low content of lauric acid (C12: 0; 2.9% of total FA). Similar
observations can be made based on our results. As in the data presented by Maertens
et al (2006b), we have also observed that among monosaturated fatty acids the
vaccenic acid (C18:1) is characterized by the greatest content, while among the
polyunsaturated ones, linoleic acid (C18:2) shows the greatest level. Rabbit milk
characterizes by 70.4% SFA, 12.8% MUFA, and 15.6% PUFA (Maertens et al., 2006b).
This means that rabbit milk contains more MUFA and PUFA than cows’ milk (55.0–
73.0% of SFA, 2.0–30.0% MUFA, and 2.4–6.3% PUFA) (2004; Mollica et al., 2021),
but more SFA and less MUFA than sows’ milk (35.27–35.26% SFA, 42.42-44.09%
MUFA, and 9.16–11.98% PUFA). We observed slightly different results in our study
compared to the data presented in the review of Maertens et al. (2006b), noting over
75% SFA, almost 13% MUFA, and over 9%. Similarly to our findings, Maertens et al.
(2006a) underline that some fatty acids were found to have a high coefficient of
variation, pointing to a high variety of their content in milk.

In our study, the percentage of different MFG changed with the day of lactation, and
as a result, it affected the mean MFG diameter, which increased with the lactation day
from 5 to almost 7 µm. Singh et al. (2006) observed just the opposite phenomenon in
cows’ milk and reported that the MFG size decreases with the progress of lactation.
According to the literature, the digestion rate of small-sized globules is greater than
that of big-sized ones (Bourlieu and Michalski, 2015). On this basis, we can speculate
that fat in rabbit milk in the first days of lactation may have a greater digestions rate
than in the late lactation phase, because of the great share of small-size MFG. The
mean from all the measurements was 5.8 µm, meaning that the MFG in rabbit milk is
bigger compared to those in cows’ milk, with an MFG of 3.2-4.5 µm, and sows’ milk
with an MFG of 2.62 µm (El-Loly, 2011; Faustini et al., 2010).

The reproductive status of rabbit does caused a variation in some of the analyzed traits,
including milk yield and chemical compounds content. We have observed a negative
impact of pregnancy on the daily milk production on day 21 of lactation, and the content
of total protein, solids-not-fat, and lactose. Research on dairy cattle also points to a
negative influence of pregnancy (late pregnancy) on milk yield and milk chemical
composition. The available research results report a negative effect of pregnancy on
milk production (Brotherstone et al., 2004; Bohmanova et al.; 2009). According to
Penasa et al. (2016), pregnancy causes a decline in milk yield and decreased content
of fat, protein, casein, and lactose in milk, because the developing fetuses need a
significant amount of nutrients and pregnancy-induced hormonal changes cause a
lower use of nutrients in milk synthesis in order to support the fetus growth (Oltenacu
et al., 1980), though the nutrient requirements for the fetus growth is much smaller than
that need for milk production, especially at the early stage of gestation. Therefore, it is
more the endocrine changes that are responsible for the effect of reproductive status,
and prolactin secretion (Xiccatto et al., 1995).

Conclusion

To conclude, we have observed that the lactation day affects the daily milk production
and the content of TS, SNF, TP, casein, lactose, C18:2, C8:3, SCC, and pH. The
percentage of small MFG decreased with lactation day, causing a possible decrease
in the digestions rates of milk. All the observed changes in milk composition over the
nursing period justify the time of solid feed introduction to rabbit kits. The reproductive
status of rabbit does caused a variation in some of the traits examined in this study.
Pregnant does produced less milk on day 21 of lactation compared to non-pregnant
ones. Moreover, we have observed a negative impact of pregnancy on the content of
total protein, solids-not-fat, and lactose in milk. Therefore, we can speculate about the
negative impact of overlapping lactations and pregnancies on rabbit kits, as their
growth is dependent on milk production and composition.

Ethics approval

According to the Journal of Laws of the Republic of Poland, Item 266, Article 1 of the
Act of 15 January 2015 on the Protection of Animals Used for Scientific and
Educational Purposes in Poland, the study does not require the ethical approval of the
Polish Ethical Commission.

Data and model availability statement

The dataset generated and analyzed during the current study is not publicly available
but is available from the corresponding author on a reasonable request.

Author ORCIDs

Agnieszka Ludwiczak 0000-0002-3009-3346

Joanna Składanowska-Baryza 0000-0002-2626-9329

Beata Kuczyńska 0000-0002-9093-9905

Ewa Sell-Kubiak 0000-0002-7642-0787

Marek Stanisz 0000-0001-8943-0266

Ewa Skrzypczak 0000-0003-3904-7925

Author contributions

Conceptualization, AL; methodology, AL ; software and validation, ESK and AL; formal
analysis, AL and JSB; resources, MS, BK, JSB, and AL; original draft preparation, AL
JSB, and ESK; review and editing, AL. All authors have read and agreed to the
published version of the manuscript.

Declaration of interest

None.

Acknowledgements

None.

Financial support statement

This research received no external funding. The article was written as part of research
conducted by the statutory funding of the Faculty of Veterinary Medicine and Animal
Science University of Life Sciences, Poland.

References

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Table 1

The number of pregnant, non-pregnant, and discarded females from Hycole rabbits.

Reproductive rhythm phases Pregnant Not pregnant Discarded*

3rd AI, n= 120

Palpation 105 15

3rd lactation 100 20 2 pregnant

4th AI, n= 98

Palpation 86 12

4th lactation n= 85 85 13 1 pregnant, 2 not pregnant

5th AI, n= 85

Palpation 80 18

5th lactation n= 98** 80 18 2 pregnant, 1 not pregnant

Abbreviations: AI = artificial insemination

*(dead/ mastitis/ other) from the total group of pregnant and not pregnant

** 85 does at 4th lactation + 13 with unsuccessful 4th AI = 98

Table 2

The chemical feed composition of two pelleted feed types (K0 and K1) provided to
Hycole female rabbits.

Items K0 K1

Total solids %1 89.73 90.78

CP %1 14.69 17.72

Fat %1 1.99 2.28

Crude fiber %1 20.44 15.29

Ash %1 6.99 7.23

ME MJ/kg 16.57 16.88

Fatty acids g/100g Fat

C8:0 0.05 0.02

C10:0 0.03 0.01

C12:0 0.22 0.18

C14:0 0.26 0.3

C15:0 0.19 0.15

C16:0 17.2 17.56

C17:0 0.17 0.15

C18:0 3.45 4.67

 C20:0 0.01 0.01

C24:0 0.45 0.38

C21:0 0.09 0.1

C14:1 0.02 0.02

C15:1 0.01 0.01

C16:1 0.36 0.34

C17:1 0.1 0.07

C18:1 70.46 69.62

C20:1 0.43 0.45

C20:2 0.28 0.16

C20:3n6 0.44 0.42

MUFA 22.05 22.62

PUFA 55.83 53.85

SFA 22.12 23.53

1Expressed as a percentage of DM.

Abbreviations: ME = metablolizable energy; MUFA = monounsaturated fatty acids;

PUFA = polyunsaturated fatty acids; SFA = saturated fatty acids
Table 3

Effect of day of lactation on the characteristics of Hycole rabbits.

Day of lactation

Items 2 9 14 21 RMSE P-value

Dam BW g 4 484b 4 753a 4 719a 4 750a 10 0.037

Litter weight g 690D 1530C 2443B 3731A 6 0.000

Litter size 9.0A 8.5B 8.2C 8.2C 0.4 0.004

Daily milk yield g 115.3D 243.9B 290.8A 205.2C 3.4 0.001

A, B, C, D (a, b)
Values within the same row with different superscripts differ significantly
at p < 0.01 (p < 0.05)

Table 4

Effect of AI effectiveness on the characteristics of the Hycole rabbits used in the

study (21st day of lactation).
 Items Pregnant Not pregnant RSME P-value

Dam BW g 4 689 4 710 10 0.651

Litter BW g 3 610b 3 836a 8 0.026

Litter weight gain g (days 2-21) 3 012b 3 226a 8 0.042

Litter size 8.2 8.3 0.5 0.718

Daily milk yield g 180.2B 221.8A 3.4 0.008

Abbreviations: AI = artificial insemination

A, B (a, b)
Values within the same row with different superscripts differ significantly at p
< 0.01 (p < 0.05)

Table 5

Pearson correlations between litter size and growth performance for pregnant and
not pregnant Hycole rabbits.

Litter weight Litter weight Litter weight gain

Items
(day 2) (day 21) (days 2-21)

Non-pregnant
 Litter size (day 2) 0.905 0.931 0.409

P= 0.0001 P= 0.0001 P= 0.115

Litter size (day 21) 0.519 0.788 0.991

P= 0.039 P= 0.001 P= 0.0001

Litter weight gain 0.638

(days 2-21) -
P= 0.008

Pregnant

Litter size (day 2) 0.969 0.931 0.103

P= 0.0001 P= 0.0001 P= 0.692

Litter size (day 21) 0.411 0.756 0.699

P= 0.108 P= 0.001 P= 0.003

Litter weight gain 0.521

(days 2-21) -
P= 0.033

Table 6

Basic physicochemical traits of Hycole rabbit milk presented by the day of lactation.
 Items Day of lactation

Day 2 Day 9 Day 14 Day 21 RMSE P-value

SCC 103 mL−1 97.44b 107.43ab 168.4a 173.9a 3.9 0.016

Casein % 7.45B 8.23A 7.72B 7.70B 0.41 0.001

TP % 10.01b 10.74a 10.26ab 10.11ab 0.40 0.017

Fat % 8.82 8.77 8.63 8.66 0.84 0.391

TS % 22.02 22.95 22.74 22.78 0.90 0.087

SFN % 14.57B 16.16A 15.16B 14.88B 0.49 0.002

Lactose % 1.98B 2.27A 2.02B 1.99B 0.17 0.000

pH 7.01A 6.89AB 6.82B 6.80B 0.14 0.000

Fat globules diameter µm 5.09Bb 5.75Ba 6.78A 7.20A 0.37 0.010

Small MFG % 57.3A 50.1AB 42.5B 40.4B 0.45 0.006

Medium MFG % 38.9B 43.6AB 48.6A 50.2A 0.42 0.003

Large MFG % 3.8B 6.3AB 8.9A 9.1A 0.46 0.001

Abbreviations: SCC = somatic cell count; TP = total protein; TS = total solids, SNF =
solids-not-fat; MFG = milk fat globules
A, B (a, b)
Values within the same row with different superscripts differ significantly at p
< 0.01 (p < 0.05).

Table 7
Effect of AI effectiveness on the basic physicochemical traits of Hycole rabbit milk
(21st day of lactation).

Items Pregnant Not pregnant RMSE P-value

SCC 103 mL−1 150.9 161.18 4.15 0.737

Casein % 8.32 8.15 0.40 0.698

TP % 11.05A 10.12B 0.39 0.008

Fat % 8.87 8.52 0.71 0.232

TS % 22.91 22.12 0.86 0.457

SNF % 16.12A 14.01B 0.52 0.002

Lactose % 2.09A 1.87B 0.14 0.003

pH 6.84 6.81 0.14 0.811

Fat globules diameter µm 5.90 5.87 0.36 0.795

Small MFG % 40.1 41.9 0.42 0.652

Medium MFG % 49.0 47.8 0.45 0.733

Large MFG % 10.9 10.3 0.41 0.897

Abbreviations: AI = artificial insemination; SCC = somatic cell count; TP = total

protein; TS = total solids, SNF = solids-not-fat; MFG = milk fat globules
A, B
Values within the same row with different superscripts differ significantly at p <
0.01
Table 8

Fatty acid profile of Hycole rabbit milk presented by the day of lactation.

Day of lactation

Items Day 2 Day 9 Day 14 Day 21 RMSE P-value

FA nmol/l 7.00A 3.39B 3.90B 3.65B 0.28 0.000

FA nmol/100g 0.60a 0.33b 0.50a 0.53a 0.28 0.035

C18:2 9.76a 9.40b 9.21b 9.00bc 0.20 0.047

C18:3 2.76a 2.42b 2.19c 2.10c 0.22 0.032

C20:4 0.63 0.63 0.63 0.63 0.10 0.811

C20:5 0.08 0.04 0.03 0.04 0.20 0.113

C22:6 0.02 0.03 0.02 0.02 0.10 0.419

C14:1 0.04 0.04 0.04 0.04 0.00 0.775

C16:1 1.30 1.32 1.27 1.21 0.14 0.105

C17:1 0.02 0.02 0.02 0.02 0.00 0.641

C18:1 7.23 7.24 7.32 7.37 0.50 0.759

C22:1 0.94 0.82 0.77 0.73 0.44 0.062

C6:0 0.47 0.48 0.45 0.43 0.14 0.290

 C8:0 32.36 30.89 32.46 31.55 0.78 0.331

C10:0 25.27 24.97 23.21 22.87 0.82 0.914

C12:0 3.31 3.33 3.30 3.31 0.33 0.328

C14:0 1.55 1.53 1.36 1.32 0.24 0.724

C15:0 0.33 0.32 0.31 0.30 0.10 0.414

C16:0 10.76 10.93 11.7 11.9 0.50 0.910

C17:0 0.34 0.34 0.35 0.35 0.00 0.851

C18:0 2.27 2.41 2.30 2.36 0.30 0.237

Total MUFA 13.25 12.55 13.14 12.78 0.47 0.235

Total PUFA 9.53a 9.29b 9.20b 9.02c 0.30 0.046

Total SFA 76.67 74.72 75.56 75.31 0.89 0.649

Abbreviations: FA = fatty acids MUFA = monounsaturated fatty acids; PUFA =

polyunsaturated fatty acids; SFA = saturated fatty acids
A, B, C (a-c)
Values within the same row with different superscripts differ significantly at p
< 0.01 (p < 0.05).

Highlights

1. How do the lactation course and the pregnancy affect rabbit milk and kits’ growth?
2. The diameter of fat globules increases from 5 to 7 µm with ongoing lactation.
3. Among the FA in rabbit milk, C18: 2 and C18: 3 are affected by lactation day.
4. Only a few milk attributes are affected by the doe’s reproductive status.
5. Results of this study may help in increasing the survival rate of rabbit kits.

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