# Reproductive robustness differs between generalist and specialist maternal rabbit lines: the role of acquisition and allocation of resources

Davi Savietto, Nicolas N.C. Friggens, J. J. Pascual

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## To cite this version:

Davi Savietto, Nicolas N.C. Friggens, J. J. Pascual. Reproductive robustness differs between generalist and specialist maternal rabbit lines: the role of acquisition and allocation of resources. Genetics Selection Evolution, 2015, 47 (2), 12 p. 10.1186/s12711-014-0073-5. http://dx.doi.org/10.1186/s12711-014-0073-5

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# Reproductive robustness differs between generalist and specialist maternal rabbit lines: the role of acquisition and allocation of resources

Savietto *et al.*
# Reproductive robustness differs between generalist and specialist maternal rabbit lines: the role of acquisition and allocation of resources

Davi Savietto<fnref n="1" />*, Nicolas C Friggens²,³ and Juan José Pascual<fnref n="1" />

**Abstract**

**Background:** Farm animals are normally selected under highly controlled, non-limiting conditions to favour the expression of their genetic potential. Selection strategies can also focus on a single trait to favour the most 'specialized' animals. Theoretically, if the environment provides enough resources, the selection strategy should not lead to changes in the interactions between life functions such as reproduction and survival. However, highly 'specialized' farm animals can be required for breeding under conditions that differ largely from selection conditions. The consequence is a degraded ability of 'specialized' animals to sustain reproduction, production and health, which leads to a reduced lifespan. This study was designed to address this issue using maternal rabbit lines. A highly specialized line with respect to numerical productivity at weaning (called V) and a generalist line that originated from females with a long reproductive life (called LP) were used to study the strategies that these lines develop to acquire and use the available resources when housed in different environments. In addition, two generations of line V, generations 16 and 36, were available simultaneously, which contributed to better understand how selection criteria applied in a specific environment changed the interplay between functions related to reproduction and survival.

**Results:** We show that, under constrained conditions, line LP has a greater capacity for resource acquisition than line V, which prevents excessive mobilization of body reserves. However, 20 generations of selection for litter size at weaning did not lead to an increased capacity of nutrient (or resource) acquisition. For the two generations of line V, the partitioning of resources between milk production, body reserves preservation or repletion or foetal growth differed.

**Conclusions:** Combining foundational and selection criteria with a specific selection environment resulted in female rabbits that had a different capacity to deal with environmental constraints. An increased robustness was considered as an emergent property of combining a multiple trait foundational criterion with a wide range of environmental conditions. Since such a strategy was successful to increase the robustness of female rabbits without impairing their productivity, there is no reason that it should not be applied in other livestock species.

**Background**

In farmed livestock, robustness (as defined by Knap [1]) represents the ability of an animal's genotype to maintain a good production level while maintaining all other life functions in a wide variety of environmental conditions (i.e. food quality, temperature, pathogen load, management, etc.). Based on this definition, robust animals, with respect to various life functions, may be considered

as 'generalist animals'. However, intensive selection of farm animals to increase productive traits has resulted in specialized breeds and strains as for example broilers [2,3] and dairy cows [4] and also for pigs [5], hens [6] and rabbits [7,8].

North American Holstein-Friesian dairy cows, an example of a highly specialized cattle breed for milk yield, prioritize milk production [9] at the expense of fertility [10]. Other examples of undesired effects in response to selection have been described for different species i.e. pigs [11,12], poultry [13,14] and rabbits [15,16], which has led to the general perception that selection degrades

<footnote>* Correspondence: dasavi@upv.es</footnote>

<footnote n="1">¹Instituto de Ciencia y Tecnología Animal (ICTA), Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
the ability of animals to simultaneously sustain produc-
tion, reproduction and health [17,18]. Nevertheless,
artificial selection of high producing animals does not
necessarily entail the emergence of negative effects as
shown for dairy cows [19] or rabbits [20,21], and breeds
and strains that can sustain production, fertility and
health under different environments (i.e. ‘generalist’) can
be obtained. This is of special interest because it indi-
cates that it is possible to select animals that are able to
balance production, reproduction and health. However,
the amount of information on the consequences of
selecting ‘generalist’ farm animals with respect to their
performance across environments, especially constrained
environments, is currently insufficient.

Theoretically, if animals are selected under non-limiting
conditions, responses to selection can be achieved without
modifying the interplay between life functions, whereas
under limiting conditions, this interplay is affected [22,23].
However, this theory is not useful to provide insights into
the consequences of selecting highly specialized animals
in relatively good environments on their ability to cope
with poor environments. Our study was designed to ad-
dress this issue, using two maternal rabbit lines: a highly
specialized line (i.e. line V) in terms of numerical product-
ivity (i.e. litter size at weaning), and a more generalist line
(i.e. line LP) that was founded for reproductive longevity
and then selected for litter size at weaning. In addition, for
line V, two generations 16 and 36, were available simultan-
eously. Lines V and LP have been shown to differ in their
ability to maintain litter size in the presence of constraints
[20,24], and also in the strategies used to attain the breed-
ing objectives (e.g. use of body reserves [25] and shape of
lactation curve [26]).

To evaluate the capacity of resource acquisition of
these lines with different selection backgrounds and
their resource allocation strategies, three environmen-
tal conditions were set up. Our aim was to study the
ability of these maternal rabbit lines to acquire (feed
intake) and allocate (litter size, milk production and
body condition) the resources available in markedly
different environments.

**Methods**

Rabbit lines and selection history

Specialist maternal rabbit line (line V)

The specialist maternal rabbit line named line V was
established at the Universitat Politècnica de València in
1981 by crossing the progeny of four specialized mater-
nal rabbit lines. After three generations of random mat-
ing by avoiding mating between close relatives, selection
to increase the number of kits weaned per litter started
[27]. Over generations, the effective population size was
maintained at 120 females and 25 males. A large number
of males were used to keep a low level of inbreeding. For

each generation, at least one male offspring per sire was
retained and the mating between relatives that shared a
grand-parent was avoided. Selection was also conducted
in non-overlapping generations of nine months. For each
generation, young females were weaned at 28 days, and
those that reached the age of 4.5 months were mated.
After parturition, mating was attempted on day 11 to
reach a reproductive cycle of 42 days, and females were
culled only after three consecutive failures due to infer-
tility. To preserve the genetic material, the Universitat
Politècnica de València rabbit selection programme has
a cryopreserved control population. Every two or three
generations of selection, embryos from a representative
sample of the best matings (for each male, two or more
straws of embryos are cryopreserved) are recovered and
vitrified. Recently, this line V reached generation 36.

Since selection began, no substantial improvement in
the selection environment of line V was made (Baselga,
personal communication). Animals are housed in flat
deck indoor cages, with free access to water and com-
mercial pelleted diets (minimum of 15 g of crude protein
per kg of dry matter (DM), 15 g of crude fibre per kg of
DM, and 10.2 MJ of digestible energy (DE) per kg of
DM). The photoperiod is set to provide 16 h of light and
8 h of dark, and the room temperature is regulated to
keep temperatures between 10°C and 28°C. Rooms are
cleaned and disinfected every week and the animals are
vaccinated against rabbit haemorrhagic diseases and
myxomatosis. Sick animals are also culled (e.g. due to re-
spiratory disorders, pasteurellosis, sore hocks, etc.). No
animals are culled for low productivity.

To evaluate the specialization process in response to
the long-term selection design, on the criterion of
reproduction only, both generations 16 and 36 of line V
were used (hereafter referred to as V16 and V36). The
parents of the V16 females used in this study, stored as
vitrified embryos, were thawed and transferred to fe-
males from another line, also selected for litter size at
weaning (line A [27,28]). After one generation without
selection, to avoid the environmental maternal effect, 72
young V16 females were obtained and compared with
79 V36 females. For detailed information on the cryo-
preservation and embryo transfer techniques used in this
study see Vicente et al. [29] and Besenfelder and Brem
[30], respectively.

Generalist maternal rabbit line (line LP)

The generalist maternal rabbit line named line LP was
established between 2002 and 2003 by applying a very
high selection intensity (i.e. two to five females in one
thousand were selected) to obtain females with a long
reproductive lifespan (i.e. at least 25 parturitions aver-
aging a minimum of 7.5 kits born alive per parturition).
To identify productive females with a long life expectancy,
three screening steps were performed in commercial
rabbit farms that were located over the whole of the
Iberian Peninsula. In the first screening, 15 females were
identified and transferred to the facilities of the Universitat
Politècnica de València. They were inseminated with
semen from males of generation 27 from line V (the
current generation in 2002). Fifteen ½ LP and ½ V males
were obtained from 12 females. These males were used to
inseminate a new set of 15 females selected from a second
screening, generating a total of 17 ¾ LP and ¼ V males.
These males were then used to inseminate a final group of
32 females from a final screening. A total of 32 males and
42 females (7/8 LP and 1/8 V) were produced from 30 fe-
males that constituted the generation 0 of line LP. From
that time on, line LP was selected to increase litter size at
weaning (currently this line has reached generation 6)
under similar conditions as those applied for line V. The
direct consequences of the multi-trait criteria used to se-
lect the founders of line LP, regardless of the environmen-
tal conditions, are that the resulting animals have a long
productive lifespan (35 days more than line V [21]), a con-
stant reproductive effort through life (the maximum re-
productive performance of line V is reached at parity four
[20]) and a better innate immune response in constrained
conditions [31,32].

Environmental conditions

To evaluate the resource acquisition capacity and re-
source allocation strategy that were derived from the
foundational criteria and selection histories of the three
genetic types, three environmental conditions were set
up by applying various room temperatures and/or diet
compositions: (1) a control environment (NC) that com-
bined normal (N) room temperatures (daily variation
from 18°C to 24°C) and a control (C) diet that was for-
mulated to achieve 11.6 MJ of DE per kg of DM, 126 g
of digestible protein per kg of DM and 169 g of acid
detergent fibre per kg of DM; (2) a heat environment
(HC) that combined high (H) room temperatures
(using a climatic chamber that was designed to pro-
duce a daily sinusoidal temperature curve from 25°C to
35°C; detailed specifications are in [33]) and the above
diet C; and (3) a nutritionally constrained environment
(NF) that combined normal room temperatures (N)
with a low-energy fibrous (F) diet that was formulated
to achieve 9.1 MJ of DE per kg of DM, 104 g of digest-
ible protein per kg of DM, and 266 g of acid detergent
fibre per kg of DM. The detailed composition of the
diets is available in [34].

Housing facilities (cages, feeders, drinkers, nest box,
etc. and their display), photoperiods (16 h of light and
8 h of dark) and reproductive management were identi-
cal for all environments.

**Experimental procedures**

At first parturition, 236 LP, V16 and V36 females were randomly allocated to one of the three environments (NC, HC, or NF) in a 3 × 3 factorial design i.e. LPNC = 26, LPHC = 31, LPNF = 28, V16NC = 22, V16HC = 31, V16NF = 19, V36NC = 25, V36HC = 29, and V36NF = 25). The number of animals initially housed under each environment depended on the availability of animals in the selection nucleus. Of these 236 females, 191 reached third parturition i.e. LPNC = 21, LPHC = 26, LPNF = 24, V16NC = 17, V16HC = 23, V16NF = 16, V36NC = 19, V36HC = 21, and V36NF = 24.

Females were subjected to a semi-intensive reproduct-
ive rhythm of 42 days and monitored until the third
parturition. They were inseminated on day 11 post-
parturition and their litters were weaned on day 28
(these are normal procedures within a selection nu-
cleus), which controlled confounding of the resource ac-
quisition of dams. Females that had not conceived on
day 11 were re-inseminated 21 days later, and this was
repeated for a maximum of three attempts; then non-
pregnant animals were culled for low fertility.

Litter size (number of kits born in total and live born kits) and litter weight (weight in g of the kits born in total and live born kits) were monitored at birth. Litters were then standardized at nine kits in the first lactation and 10 kits in the second lactation, so that the three genetic types shared a similar lactation burden. Subsequently, dead kits were not replaced, and both litter size and weight were monitored at weaning. Milk yield (g/d) was measured four days a week during the first three weeks of lactation by weighing females before and after nursing. During the whole experiment, females were fed *ad libitum*. Dry matter intake was measured weekly during the first three weeks of lactation, and during the weaning to parturition interval, which varied according to the females' real reproductive rhythm. The females' digestible energy intake (MJ/d) was calculated based on DM intake and apparent digestible coefficients of gross energy that were obtained in a digestibility trial for LP, V16, and V36 females under the NC, HC, or NF environments (values available in [34]).

Female body condition was assessed by measuring live
weight (at 0, 7, 14, 21 and 28 days post-parturition) and
perirenal fat thickness (PFT) (at 0, 14 and 28 days post-
parturition). PFT (mm) was measured by ultrasonog-
raphy according to Pascual et al. [35].

**Data management and statistical analyses**

The data used in this work has already been partially
used elsewhere [25,26,34]. To avoid any influence from
non-controlled factors that may affect resource acquisi-
tion capacity and/or resource allocation of the three gen-
etic types, data for 45 of the 236 housed females that did
not reach third parturition were discarded. Reasons for culling 17 females were low fertility (LPHC = 2, LPNF = 1, V16NC = 1, V16NF = 1 and V36NC = 1), retained foetus (LPNC = 1 and V36NC = 1) or diseases (i.e. pasteurellosis: LPNF = 2, V16HC = 2, V16NC = 2 and V16NF = 2 and colibacillosis: V16HC = 1). Another 28 females were found dead (LPHC = 3, LPNC = 3, LPNF = 2, V16HC = 5, V16NC = 2, V36HC = 8, V36NC = 4, and V36NF = 1).

Prolificacy of LP, V16 and V36 females was assessed, under each environment, as the cumulative number of kits produced during the second and third parturitions. Since litters were standardized at birth, the cumulative number and the average weight of weaned kits represented, to some extent, the female's maternal ability. The resource acquisition capacity was measured as the total DM or DE intake during the first three weeks of lactation and during the period between weaning and parturition (regardless of total intake and number of days between weaning and parturition; i.e. we considered the real weaning to parturition interval of each female) during the first two reproductive cycles. Female live weight and PFT are the average values measured during the first two reproductive cycles (i.e. between first and second parturitions and between second and third parturitions).

Statistical analyses were performed using the general linear model function of R software [36] and the least square means were computed using the lsmeans package [37]. The model used to analyse the cumulative numbers of kits born in total, live born kits, and weaned kits, as well as the individual weight of kit weaned included an effect for environment (E: HC, NC and NF), genetic type (G: LP, V16 and V36) and the interaction between both:

$$Y_{ij} = E_i + G_j + (E_i \times G_j) + e_{ij}. \quad (1)$$

The model used to analyse the weight of individual kits (for kits born in total and live born kits) also included the total number of kits born (KT) as a covariate:

$$Y_{ijk} = E_i + G_j + (E_i \times G_j) + KT_k + e_{ijk}. \quad (2)$$

The model used to analyse intakes of DM and DE, live weights and PFT included parturition order (PO: first or second) as a fixed effect:

$$Y_{ijk} = E_i + G_j + (E_i \times G_j) + PO_k + e_{ijk}. \quad (3)$$

Finally, the model used to analyse milk yield incorporated the average number of kits during lactation (KL) as a covariate:

$$Y_{ijk} = E_i + G_j + (E_i \times G_j) + KL_k + e_{ijk}. \quad (4)$$

Reaction norms were used to evaluate the overall environmental sensitivity according to foundational criteria or selection histories (Figures 1, 2, 3 and 4). Data on the

percentage of energy acquired that was allocated to milk synthesis, litter production (stillborn kits and live born kits) and body growth are presented in a radial plot (Figure 5) to compare the different allocation strategies used by each genetic type under the different environmental constraints. For each female, energy acquired (MJ) was calculated as the sum of energy intake during the first three weeks of lactations one and two (excluding data of the fourth week) plus the energy intake recorded during the weaning to parturition intervals. The amount of energy in milk was calculated as the total amount of milk produced in the first three weeks of lactations one and two assuming that the energy content of milk was 8.5 MJ per kg of milk [38]. Body energy was calculated as the cumulative loss of body energy during lactations one and two based on carcass energy content at parturition and at weaning that was estimated with the equation developed by Pascual et al. [35]:

$$\text{Body} = 2.51 + 0.012 \times \text{LW} + 0.00018 \times \text{PFT}^3, \quad (5)$$

where Body is the estimated body energy (MJ), LW is the live weight (g) and PFT is the perirenal fat thickness (mm). Finally, energy content of the litter was calculated as the total weight of stillborn and live born kits in the second and third parturitions assuming 3.4 MJ per kg of kits produced [39]. Plotted values are lsmeans of a statistical model equal to model (1).

## Results

### Resource acquisition capacity

Average daily DM and DE intakes of LP, V16 and V36 females under normal (NC), heat (HC), and nutritional (NF) challenging environments are in Figures 1A and 1B, respectively. As expected, NF was associated with an increased DM intake but a limited DE intake, whereas HC depressed both DM and DE intakes. Performance results revealed that LP, V16, and V36 females responded differently to the constrained conditions. Under the HC and NF environments, LP females displayed a greater resource acquisition capacity (as shown by energy intake; P <0.05) than both V16 and V36 females. Under normal conditions, DM intake was not significantly different between LP, V16 and V36 females. However, LP females were able to ingest on average 0.27 MJ of DE/d more than V females (P <0.05) under the HC environment. Under the NF environment, LP females had a higher DM intake than V16 and V36 females (on average +33.9 g DM/d; P <0.05), which resulted in LP females having a similar DE intake to that observed in normal conditions. The decrease in DE intake due to exposure to the nutritional challenge was higher for V16 (–0.28 MJ DE/d compared to V16 under the NC environment; P <0.05) than for V36
A. Dry Matter Intake

<figure><img src="image_4.png" /></figure>

B. Digestible Energy Intake

<figure><img src="image_5.png" /></figure>

**Figure 1** Resource acquisition capacity of LP, V16 and V36 females housed under different challenging conditions. Average daily dry matter (g/d) and digestible energy (MJ/d) intakes from first to third parturition (except from day 21 to day 28 of lactation) are in panels A and B, respectively. Blue: LP, orange: V16 and red: V36; heat (HC), normal (NC) and nutritional (NF) challenging conditions; vertical lines represent the standard error of means.

females (–0.19 MJ DE/d compared to V36 females under the NC environment; P = 0.17).

Resource allocation strategies

At first parturition, LP, V16 and V36 females had on average a total number of kits born equal to 9.3, 9.5 and 10.3, respectively, with 8.6, 8.4 and 9.3 live born kits, respectively. There were no statistically significant differences between the lines (the pooled standard error of means was 0.57 for total number of kits born and 0.68 for number of live born kits).

Constrained conditions did not affect the reproductive performance of LP females (Figures 2A and 2B), which had similar litter sizes at birth under all environments (e.g. 10.4, 9.9 and 9.9 total kits born per parturition under the NC, HC and NF environments). However, the constrained environmental conditions affected the reproductive performance of V females and this effect differed between generations 16 and 36. Under the HC and

NF environments, V16 females had a much lower total number of kits born (–2.6 and –3.2 kits per parturition under HC and NF, respectively; P <0.01) compared to the NC environment but the decrease in number of live born kits was less pronounced (–1.6 and –1.7 kits per parturition under HC and NF, respectively; P = 0.67). In contrast, under the HC and NF environments, V36 females were more able to maintain the total number of kits born (–2.0 and –1.4 kits per parturition under HC and NF, respectively; P = 0.35) than the number of live born kits (–3.4 kits per parturition; P = 0.003 and –2.1 kits per parturition; P = 0.24 under HC and NF, respectively). After litter standardization at birth, no difference in the number of kits weaned was observed between genetic type or environmental constraint (on average 9.5 kits per parturition; Figure 2C).

Reaction norm plots for average individual weights of kits at birth and weaning are in Figure 3. Productive females with a long life expectancy gave birth to kits with

A. Number of Kits Born in Total

<figure><img src="image_6.png" /></figure>

B. Number of Live Born Kits

<figure><img src="image_7.png" /></figure>

C. Number of Weaned Kits

<figure><img src="image_8.png" /></figure>

**Figure 2** Litter sizes of LP, V16 and V36 females housed under different challenging conditions. Cumulative number of kits born in total and number of live born kits in the second and third parturition are in panels A and B, respectively. Cumulative number of kits weaned during the first two lactations is also shown C. At birth, litters were standardized at 9 kits in the first lactation and at 10 kits in the second lactation (true value: 18.9 ± 0.7 kits per female; mean ± s.d.) with dead kits not replaced. Blue: LP, orange: V16 and red: V36; heat (HC), normal (NC) and nutritional (NF) challenging conditions; vertical lines represent the standard error of means.
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 3** Weight of litters of LP, V16 and V36 females housed under different challenging conditions. Average individual weights (g) of kits born in total, live born kits and weaned kits are in panels A, B, and C, respectively. Blue: LP, orange: V16, and red: V36; heat (HC), normal (NC) and nutritional (NF) challenging conditions; vertical lines represent the standard error of means.</figcaption></figure>

a lower weight under constrained conditions than under normal conditions (on average -6.9 and -4.7 g for total kits born and live born kit, respectively; P <0.05). The individual weights at birth of V16 kits under constrained and normal conditions were not significantly different. However, although the individual weights of V36 kits at birth were similar under NF and NC environments, they were significantly smaller under the HC environment (-6.0 and -6.5 g for total kits born and live born kits, respectively; P <0.05). Across environments, the individual weight of kits weaned by LP females was on average 26.7 g greater than that of kits weaned by V16 and V36 females (Figure 3C).

Average live weight, PFT and daily milk yield of fe-
males are in Figure 4. Only V16 females displayed a sig-
nificant decrease in live weight under constrained
conditions (-200 and -250 g under HC and NF com-
pared to NC, respectively; P <0.05). Response in PFT to
the different environments varied between genetic types.
Although PFT was similar for all genetic types under
NC and NF environments, LP females showed a greater
decrease in PFT under the HC environment than under
the NC environment (-0.28 mm; P <0.05). Regardless of

environmental constraints, LP females produced more
milk than V16 and V36 females (on average +21.1 g/d).
Constrained conditions resulted in lower milk yields
compared to normal conditions for both LP (-34.0
and -29.9 g/d under HC and NF, respectively; P <0.001)
and V36 females (-38.0 and -33.1 g/d under HC and NF,
respectively; P <0.001). In contrast, V16 females had simi-
lar milk yields under all environmental conditions.

**Discussion**

Selection context and origin of maternal rabbit lines

The context in which the founder LP females originated
and the selection criteria and environmental conditions
applied to select line V are important to understand the
distinct resource acquisition capacities and allocation
strategies used by these lines to attain their fitness. It is
also important to note that generally, under commercial
conditions, healthiness and prolificacy (i.e. fertility, litter
size and maternal ability) are the main factors that con-
dition female fitness and lifespan.

The LP line was established by selecting females from
different commercial farms in the Iberian Peninsula that
had at least 25 parturitions with a minimum number of

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 4** Body condition and milk yield of LP, V16 and V36 females housed under different challenging conditions. Average live weight (kg), perirenal fat thickness (mm), and daily milk yield (g/d in the first three weeks) of the first two reproductive cycles are in panels A, B, and C, respectively. Blue: LP, orange: V16 and red: V36; heat (HC), normal (NC) and nutritional (NF) challenging conditions; vertical lines represent the standard error of means.</figcaption></figure>
A. Normal Challenge

<figure><img src="image_15.png" /></figure>

B. Heat Challenge

<figure><img src="image_16.png" /></figure>

C. Nutritional Challenge

<figure><img src="image_17.png" /><figcaption>**Figure 5** Radial plots showing the percentage of total energy acquired and assigned to milk production, litter and body.</figcaption></figure>

Data on milk (MJ), litter (MJ of stillborn and live born kits), and body energy (MJ) of LP (blue area), V16 (orange lines), and V36 (red lines) females housed under normal, heat or nutritional challenging conditions are in panels A, B, and C, respectively. Energy content of milk was calculated assuming 8.5 MJ per kg of milk [38]. Energy content per kg of kit was assumed to be 3.4 MJ [39] and energy content in the body (MJ) was calculated from equation (5) developed by Pascual et al. [35]. The scale used for milk energy is different from the other axis (ranging from 20 to 32%). The average standard error of the means for each component trait was: 0.05 for Body, 1.16 for Milk, 0.10 for Live Born and 0.04 for Stillborn. The standard error of means for the different lines and environments are in Figures 1, 2, 3 and 4.

7.5 live born kits per parturition [21], which led to very robust females [20,24,25,31,32]. This robustness is the consequence of the seasonal and punctual fluctuations in the environmental conditions that occur in commercial farms. In order to avoid culling and ensure a long productive lifespan, females must have a good long-term adaptive capacity to adequately face these environmental constraints across many parturitions. Here, we also observed that LP females displayed greater reproductive stability under constrained environments (i.e. litter size was maintained) than V16 and V36 females, which is why they are perceived as generalist animals.

Line V was founded 30 years ago from four specialized maternal rabbit lines [27] and, ever since, has been selected to increase litter size at weaning (it has now reached generation 36) in a context where the intergenerational change (9 months) limited the females' reproductive life and the time span during which they demonstrate their genetic potential. In addition, the interval between parturitions of 42 days that was used (females are mated on day 11 post-parturition), with litters weaned on day 28 post-parturition, places selection pressure on females to adequately nurse the current litter while the future litter is developing in utero. In this sense, V females have been selected to cope with the short-term nutritional stress involved in weaning the greatest number of kits possible. Therefore, during the selection process, V females must have adopted specific strategies to be selected. Our study shows that the resource usage patterns to sustain litter size at weaning differ between the two generations of line V i.e. V16 and V36, which indicates that a specialization process is ongoing.

Resource acquisition capacity

The constrained conditions of HC and NF environments were designed to limit the energy intake of rabbit females. Hot temperatures impair energy intake; i.e. even when fed with a high-energy-density diet, the animals
are unable to meet their daily requirements [40]. High-
fibrous low-energy diet act differently; females increase
their feed intake in an attempt to satisfy their daily en-
ergy requirements but are physically limited [41]. The
designed constraints worked as expected.

As hypothesized, the resource acquisition capacity of rabbit females differed depending on foundational criteria and selection histories of the lines. LP females displayed a greater resource acquisition capacity than V females (regardless of the generation of selection), but only in highly demanding conditions (HC and NF). This pattern was already observed by Theilgaard et al. [24] for LP and V females. Conversely, 20 generations of selection to increase litter size at weaning was not accompanied by an overall increase in the female's resource acquisition capacity. Selection programmes using crossbred rabbit females also showed that overall feed intake did not increase [42], although Quevedo et al. [43] and Savietto et al. [26] observed a different time-trend in the feed intake of crossbred and pure rabbit females from recent generations of selection, i.e. they had a higher intake during early lactation. This strategy combined with adequate management of body reserves is adapted to the hot summers of Mediterranean areas, where selection of line V is carried out. Moreover, Savietto et al. [26] reported that under HC and NF constrained conditions, V16 and V36 females presented a similar time-trend for DE intake and a similar resource acquisition capacity. Therefore, 20 generations of selection for litter size at weaning did not increase the environmental sensitivity of rabbit females with respect to resource acquisition capacity.

The higher resource acquisition capacity of LP females under challenging conditions seems to be the main strategy used by the founder females of this line to achieve a long and productive lifespan. In commercial farms, long productive lifespan is only attained by females that maintain their prolificacy (adequate litter size at birth and at weaning) over different reproductive cycles. In commercial farms, food is freely available throughout the females' productive life. However, to meet their needs and adequately face any possible environmental constraints, females can develop different strategies. Accretion of body reserves is one possibility whereby females accumulate fat in non-limiting conditions to use it when the acquisition of resources is limited (e.g. hot seasons, poor food quality or even pathogens). This is a well-known strategy used by different farmed livestock species, to alleviate periods of nutritional stress (e.g. cows [44] or rabbits [18]). However, this strategy is probably not the most suitable to maximize fitness, since during their productive life, rabbit females may encounter expected but also unexpected constraints of different intensities, durations, and even frequencies. In addition,

under conditions of unlimited food availability, females
may gain fat and carrying excess body fat is costly, both
in evolutionary [45] and metabolic terms [46]; indeed,
some surveys have shown that excess body fat in rabbit
females can be associated with an increased risk of being
culled [47,48]. Bearing this in mind, the high resource
acquisition capacity observed for LP females seems to be
a more reasonable strategy to alleviate the negative ef-
fects of limiting conditions since it prevents excessive
mobilization of body reserves, and thus favours a long
productive lifespan. However, for this resource acquisi-
tion strategy to be effective, it needs to be associated
with an adapted allocation of the resources among the
competing life functions.

Resource allocation strategies

Neither the different foundational criteria of LP and V
females (i.e. LP vs. V36) nor the selection history of line
V (i.e. V16 vs. V36) altered the females’ resource acquisi-
tion capacity in normal conditions. However, allocation
of the resources differed. Figure 5A represents the per-
centage of energy acquired and allocated to different life
functions during two consecutive reproductive cycles
under the NC environment. We observed that, in pro-
ductive females with a long life expectancy, resources
are not wasted; instead a greater proportion of the ob-
tained resources are assigned to produce milk, which is
very important to ensure survival and development of
the new-born kits [49], and for body reserves but at an
adequate level to avoid the risks related with an excess
of body fatness [45]. Moreover, only a very small amount
of energy was allocated to stillborn kits, which is an in-
teresting strategy to avoid risks related with late gesta-
tional loss [50].

After 20 generations of selection to increase litter size
at weaning, V36 females were able to assign 0.155 MJ
per parturition more than V16 females to produce viable
kits (live born kits), thus reducing the expenses to non-
viable kits (stillborn). García and Baselga [8] already re-
ported an increased number of live born kits in response
to selection for line V, and Quevedo et al. [43] observed
a greater efficiency of DE use for foetal growth. Selection
to increase litter size at weaning was also accompanied
by higher milk yield in early lactation [26,43], which rep-
resents a critical period for the survival of kits [49].
These strategies had no negative impact on body fat re-
serves [43,47] through lactation [26]. However, there is
evidence of a trade-off between the pregnant uterus and
the mammary gland when rabbit females are concur-
rently pregnant and lactating [51]. In a selection context
where fitness is attained by females that wean large lit-
ters in short intervals, selection of line V seems to favour
females that can rapidly change the priority between the
current (kits being nursed) and future litters (foetus
development). In addition, in a selection design where the time-span during which females can show their genetic potential is limited (selection takes into account repeated measures on the same animal [27]) and that promotes different strategies to allocate the obtained resources, maintaining reproduction depended more on the accretion and use of body reserves [26].

An interesting aspect of our study is that the differences in adaptive capacities that the different lines have acquired and that contribute greatly to their robustness, were only revealed by comparing the lines under different environmental conditions. Understanding the strategy adopted by LP females to achieve a long productive lifespan was only possible by evaluating the females' responses in constrained conditions. Similarly, the emergence of environmental sensitivity in response to selection, which is indicative of specialization, could only be observed under constrained conditions. The differences in life history strategies between LP and V36 females were highlighted under the HC environment. Previously, it was shown that LP females used body reserves to maintain their reproductive performance in the presence of constraints [20]. However, our results show that LP females also made use of their greater resource acquisition capacity to sustain reproductive performance. Intriguingly, this increased resource acquisition capacity was coupled with an adjustment in the allocation of these resources such that the investment in litter size was limited. Although LP females acquired more DE than V36 females, they did not invest the energy surplus in a larger litter size (see Figure 5B). They seem to be able to avoid the follow-on risks related with intense foetal growth by partitioning relatively less energy to produce non-viable kits than V36 females (e.g. LP females assigned only 8.5% of the 1.6 MJ used for litter production to produce stillborn kits while V36 females assigned 17.3% of the 1.5 MJ used for litter production to produce stillborn kits). Instead, the greater DE intake capacity was partitioned into milk yield, which ensured the survival of kits, without reserving additional resources to maintain the fat level to values similar to those observed under the NC environment. For LP females, it seems that fat reserves are a safety factor, rather than a necessary energy surplus to ensure reproduction [25].

The advantage obtained by V36 females in response to selection for litter size after 20 generations was observed under constrained conditions, especially under the HC environment. V36 females reduced milk yield and safeguarded body condition (i.e. live weight and PFT), which led to a small decrease in total number of kits born (-1.9 kits per parturition compared to the NC environment). However, the number of live born kits was smaller under constrained conditions compared to normal conditions (-3.4 kits per parturition). In contrast,

under constrained environments, V16 females maintained milk yield to levels similar to those under the NC environment, which increased the relative partition to the current litter. V16 females also displayed a decreased gestational effort (i.e. -2.6 kits born in total per parturition, compared to the NC environment) which means that the available resources were adjusted to give birth to viable kits. Contrary to what was observed under the HC environment, V36 females under the NF environment partitioned the energy resource that was not aimed at producing milk into gestation to ensure a small reduction in litter size (-1.4 total kits born per parturition compared to the NC environment) but more viable kits (+1.3 live born kits per parturition).

Our work provides evidence of changes in the resource acquisition capacity and the allocation of obtained resources into different life functions as a result of different selection criteria in two maternal rabbit lines. Line LP was founded by selecting females with a long productive lifespan from commercial farms [21] where culling and early mortality are the main factors that limit lifespan. A long productive life is, thus, an indirect indicator of successful adaptation to variations in environmental conditions (for other examples see [52,53]). Reproduction has a cost [54] and there is a consensus on the existence of a trade-off between reproduction and lifespan [55]. However, trade-offs also depend on the environmental conditions [23,55], thus the selection of species or lines that can obtain and partition resources into competing functions depends on the interaction between genotype and environment [23]. Our work demonstrates that this is the case for LP females. They did not prioritize a single function, but had an increased acquisition capacity,

Relative Priorities Between Actual and Future Litters

<figure><img src="image_18.png" /></figure>

**Figure 6 Relative priorities between the current and future litters in rabbit females.** Scheme representing the changes in the relative priorities between the litter being nursed (dotted lines) and the litter under gestation (solid lines) for V16 (orange) and V36 (red) females; two consecutive cycles are represented. Twenty generations of selection for litter size at weaning resulted in females that are able to change priorities rapidly between the current and future litters (reprinted with permission of Pascual et al. [18]).
allocating resources to sustain prolificacy without increas-
ing excessive mobilization of body reserves. In this case,
fat reserves are a safety factor.

Selection to increase litter size at weaning over 20 gen-
erations did not favour a greater acquisition capacity of
female rabbits, but the selection criteria were attained by
female rabbits that proportionally assigned more re-
sources to favour litter size at weaning. The main reason
for not favouring a greater resource acquisition capacity
seems to be related to the change in priorities between
the current and future litters, as suggested by [45]. Sev-
eral arguments support this hypothesis i.e.: the limited
time span during which females can express their gen-
etic potential together with the evidence available from
the literature on the competition between the gravid
uterus and the mammary gland [51], the larger litter size
at birth with a relatively high milk yield during early lac-
tation and low milk yield during late lactation [26,43]
and the greater dependence of V36 females on fat re-
serves to ensure reproduction [26]. A schematic repre-
sentation of the hypothesis that V36 females allocated
resources to functions related to the rapid change in pri-
ority between litters, rather than to increasing resource
acquisition, is in Figure 6 (reprinted from Pascual et al.
[18]). Additional research on how time limitations affect
the temporal pattern of the allocation of resources
(under non-limiting conditions) between life functions is
necessary.

Ethical statement

The Research Ethical Committee of the Universitat de
Politècnica de València gave ethical approval for the pro-
tocols of this research.

Competing interests

The authors declare that they have no competing interests.

Author's contributions

DS, NF and JP participated in the experimental design, interpretation of data and writing of the article; DS and JP carried out data collection and DS performed the statistical analyses and prepared the figures. All authors read and approved the final manuscript.

Acknowledgements

The authors thank Juan Carlos Moreno Pardo, Luis Ródenas Martínez and Eugenio Martínez Paredes for their technical support, and Doctors Manuel Baselga Izquierdo, Enrique Blas Ferrer and Concha Cervera Fras for their valuable comments in the first version of this work. We also thank the Spanish Ministry of Education and Science (Project AGL2011-30170-C02-01) for the economic support.

Author details

¹Instituto de Ciencia y Tecnología Animal (ICTA), Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain. ²INRA, UMR 791 Modélisation Systémique Appliquée aux Ruminants (MoSAR), 16 rue Claude Bernard, F-75231 Paris, France. ³AgroParisTech, UMR 791 Modélisation Systémique Appliquée aux Ruminants (MoSAR), 16 rue Claude Bernard, F-75231 Paris, France.

Received: 20 February 2014 Accepted: 14 October 2014

Published online: 17 January 2015

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