Light is a vital indicator for animals to perceive environmental changes and can
cause changes in the reproductive cycle according to Webb [1]. Chang et al. [2],
Chellappa et al. [3], Chang et al. [4], van der Lely et al. [5], and Rahman et al. [6]
showed that the effect of light intensity and duration on the physiology and
behaviour of mammals varied with light intensity and duration. According to
Mattaraia et al. [7], increasing the duration
of light has a positive effect on the reproductive success of female rabbits; the
rabbit acceptance rate is greater under a 16 L:8 D photoperiod than under an 8 L:16
D photoperiod. Furthermore, whereas it is usual practice in European rabbit
production farms to expose breeding females to artificial light for 15 to 16 hours
each day throughout the year, many rabbit farms in China have adopted this lighting
scheme of time. However, as studies have progressed, the colour of light has begun
to be considered [8]. Colour, according to
Bourgin et al. [9], plays a more essential and
nuanced function than previously assumed and is a critical element to consider.
Despite the relevance of light colour for commercial rabbits maintained in buildings
with artificial light, few studies have described the influence of light colour on
rabbit reproduction. Because the number of rabbits in China is currently quite high,
rabbit farms in China employ the lighting system used in Europe. Due to geographical
variations, it is vital to investigate whether there is a better suitable lighting
plan for Chinese rabbit farms.Furthermore, compared to research on the influence of light colour on avian
reproductive performance [10,11], there have been comparatively few
investigations on the molecular mechanism by which light colour may alter rabbit
reproductive performance and follicular growth. Gonadotropin releasing hormone
receptor (GNRHR), follicle stimulating hormone receptor (FSHR), and luteinizing
hormone receptor (LHR) are three key candidate genes for mammalian reproductive
characteristics. GNRHR is expressed in the brain, pituitary gland, and ovaries, as
demonstrated by Zerani et al. [12], and
rabbit oocytes, granulosa cells (GC), membrane cells, and zona pellucidae express
particular GnRH receptors. FSH and LH both play key roles in the growth and
maturation of follicles, and both the FSHR and LHR proteins are expressed in rabbit
zona. According to the findings, the GNRHR, FSHR, and LHR genes all have a role in
rabbit reproduction. The dynamic processes of ovarian follicle growth and atresia in
female animals are intimately associated with GC death throughout the whole oestrus
cycle [13,14], and GC apoptosis is mediated by the BCL-2 protein family [15,16].
B-cell lymphom (BCL)-2, a crucial apoptosis-related gene, can prevent cells from
undergoing additional apoptosis by blocking the ultimate apoptosis process. BAX, a
member of the BCL-2 family, has the inverse effect and can accelerate apoptosis.
Ovarian follicle formation is the cornerstone of female mammalian reproduction. The
proliferation of GCs is an important mechanism essential for optimal follicular
development. Adiguzel et al. [17], Li et al.
[18] demonstrated that GC cycle arrest at
the G0/G1 phase is highly correlated with pathways associated with stress and FOXO
signalling and that the increased proportion of GCs at the arrested G0/G1 phase was
accompanied by increased FOXO1 expression in vivo and in
vitro. In this work, we chose these genes with well-defined roles and
investigated the effects of light colour on reproduction and follicular development
gene expression in rabbits to see if there is a better lighting system for rabbit
breeding facilities than the one now in use. Our work serves as a foundation for
future research on the effects of light and colour on female reproductive
health.
MATERIALS AND METHODS
Experimental animals and experimental design
Female New Zealand rabbits were obtained from the Liu He Animal Science Base
(118°36′E, 32°29′N), Jiangsu Academy of Agricultural
Sciences, China (experimental rabbit production licence number: SCXK
(SU)2017-0008). The Care and Use of Laboratory Animals (Ministry of Science and
Technology of the People’s Republic of China) and the Animal Care and Use
Committee of Yangzhou University, Yangzhou, China (licence number:
SYXK(SU)2017-0044) authorized all procedures. The 5-month-old nulliparous New
Zealand rabbits (total of 1,068) weighed 3.6–4.4 kg live body weight. All
rabbits were randomly assigned to one of four groups and confined to wire mesh
cages (600 × 500 × 400 mm) with red light-emitting diode (LED)
light (660–700 nm, Group R), green LED light (500–560 nm, Group
G), blue LED light (440–480 nm, Group B), or white LED light
(400–700 nm, Control Group W). The illumination was given by various
coloured LED strip lights placed in the centre of the cage ceiling. The light
intensity was evaluated in the cage’s centre using a Spectronics (SP)
XRP-3000 photometer (Spectronics Corporation, Melville, NY, USA), and the
measured values were 150 ± 3.0 lx, 150 ± 2.8 lx, 150 ± 3.1
lx, and 150 3.2 lx, indicating that the intensity was similar for each group.
The lighting schedule was 16 L:8 D-15 d/6:00 am–22:00 pm (3 days before
artificial insemination (AI) to 12 days after AI) (Fig. 1).
Fig. 1.
Schematic diagram of the lighting schedule.
AI was performed on day 7 of the experiment. Pregnancy diagnosis was
performed by palpation on day 19 of the experiment (12 d
postinsemination). Kindling was performed on day 38 of the experiment,
and weaning was performed on day 73 of the experiment (the kits were
weaned at 35 d of age). The lighting schedule was 16 L:8 D-15 d/6:00
am–22:00 pm (3 d before AI to 12 d post-AI). LED, light-emitting
diode; AI, artificial insemination.
Schematic diagram of the lighting schedule.
AI was performed on day 7 of the experiment. Pregnancy diagnosis was
performed by palpation on day 19 of the experiment (12 d
postinsemination). Kindling was performed on day 38 of the experiment,
and weaning was performed on day 73 of the experiment (the kits were
weaned at 35 d of age). The lighting schedule was 16 L:8 D-15 d/6:00
am–22:00 pm (3 d before AI to 12 d post-AI). LED, light-emitting
diode; AI, artificial insemination.During the studies, the rabbits were kept in a closed and ventilated facility
with a maximum temperature of 26°C, a minimum temperature of 22°C,
and a relative humidity of 50% to 60%. All cages included food and drinking
faucets. Water from the nipple drinkers and a commercial feed (digestible
energy: 11.2 MJ/kg, crude protein: 186 g/kg, crude fibre: 155 g/kg) were freely
accessible.On day 7, AI was conducted, and ovulation was stimulated with an intramuscular
injection of LHR-A3 (1 g, Ningbo Second Hormone Factory, Zhejiang, China).
Palpation was used to diagnose pregnancy on day 19 of the trial (12 d
postinsemination). Then, on day 76 of the trial, AI was conducted, and ovulation
was induced by intramuscular injection of LHR-A3 (1 g, Ningbo Second Hormone
Factory). On day 88 of the trial, pregnancy was determined by palpation (12-d
postinsemination). The experiment was Kindled on day 107 and weaned on day 142.
(The kits were weaned at 35 d of age). The lighting schedule was as follows: 16
L:8 D-15 d/6:00 am–22:00 pm (3 d before AI to 12 d post-AI). On day 145
of the trial, slaughter was carried out (Fig.
2). The conception rate was estimated by dividing the total number of
inseminated does by 100. The total litter size, live litter size, and litter
weight were measured on the day of kindling, and the kindling rate (the number
of kindled does multiplied by the number of inseminated does multiplied by 100)
was computed. Following delivery, the litters were divided into groups based on
the average number of living kits (maximum 8 kits). Preweaning mortality was
estimated as the number of kits deceased at weaning divided by the total number
of kits born alive multiplied by 100 on the day of weaning (35 d
postparturition). At 35 days old, the kittens were weaned.
Fig. 2.
Slaughter schedule.
AI was performed on day 76 of the experiment. Pregnancy diagnosis was
performed by palpation on day 88 of the experiment (12 d
postinsemination). Kindling was performed on day 107 of the experiment,
and weaning was performed on day 142 of the experiment (the kits were
weaned at 35 d of age). The lighting schedule was 16 L:8 D-15 d/6:00
am–22:00 pm (3 d before AI to 12 d post-AI). Slaughter was
performed on day 145 of the experiment. The coloured horizontal bars
(red, green, blue, white) indicate a 16 L:8 D photoperiod; the
grey-outlined horizontal bars (white) indicate a 12 L:12 D photoperiod.
AI, artificial insemination, LED, light-emitting diode.
Slaughter schedule.
AI was performed on day 76 of the experiment. Pregnancy diagnosis was
performed by palpation on day 88 of the experiment (12 d
postinsemination). Kindling was performed on day 107 of the experiment,
and weaning was performed on day 142 of the experiment (the kits were
weaned at 35 d of age). The lighting schedule was 16 L:8 D-15 d/6:00
am–22:00 pm (3 d before AI to 12 d post-AI). Slaughter was
performed on day 145 of the experiment. The coloured horizontal bars
(red, green, blue, white) indicate a 16 L:8 D photoperiod; the
grey-outlined horizontal bars (white) indicate a 12 L:12 D photoperiod.
AI, artificial insemination, LED, light-emitting diode.On the 145th day following the LED light treatment, 15 female rabbits with
similar body weights and at the same physiological stage (marked by a vulva
colour of dark red or purple) were chosen for slaughter in each group of
experimental rabbits. The right ovarian tissues were immediately snap-frozen in
liquid nitrogen and kept at −80°C until the relative quantity of
mRNA was determined. The ovarian tissues were dissected free of fat and
mesentery and placed in 4% paraformaldehyde.
Experimental methods
Histological evaluation
The ovarian samples were preserved in 4% formalin solution (Sigma-Aldrich,
St. Louis, MO, USA) for 24 hours immediately after the rabbits were killed.
They were subsequently treated using normal histological procedures before
being embedded in paraffin blocks with a Leica EG 1150H paraffin embedding
station (Leica Microsystems, Wetzlar, Germany) [19,20]. Each
sample was sliced into three- to five-mm-thick slices with a microtome
(Leica RM2255, Leica Microsystems) and placed on standard glass slides.
Haematoxylin and eosin were used to stain the slices. Primary and secondary
ovarian follicles, as well as large nonovulated haemorrhagic follicles, were
identified, classified, and counted. All follicles were measured twice, and
only those with clearly visible oocytes were counted.
RNA isolation and real-time reverse transcription polymerase chain
reaction
Total RNA was isolated from ovarian samples using TRIzol reagent (Invitrogen,
Carlsbad, CA, USA) according to the manufacturer’s procedure. The
optical density at 260 and 280 nm was used to calculate the quantity and
purity of total RNA, and 1.8% agarose gel electrophoresis was used to
determine RNA integrity. After that, 1.5 g of RNA was processed with DNase
before being reverse-transcribed into cDNA with a
PrimeScript® RT Reagent Kit (Takara, Dalian, China).
The cDNA samples were kept at −80°C.RT–PCR examination of the relative abundance of GNRHR, SHR, LHR, Bcl,
BAX, FOXO1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in ovarian
samples was performed using an Applied Biosystems 7500 Real Time PCR System
and SYBR Premix Ex Taq Kit (Applied Biosystems, Carlsbad, CA, USA) (Takara,
Dalian, China). Each PCR mixture contained 20 L of cDNA, 10 L of SYBR Premix
Ex Taq, 0.5 L of forward PCR primer, 0.5 L of reverse PCR primer, 0.5 L of
ROX Reference Dye II, and 6.5 L of ddH2O.Rabbit mRNA sequences for GAPDH, GNRHR, FSHR, LHR, BCL-2, BAX, and FOXO1 were
acquired from NCBI GenBank. Primer Premier 5.0 software was used to create
the primers. Table 1 shows the
sequence, GenBank number, and length of each primer set.
bp, base pair; GAPDH, glyceraldehyde 3-phosphate dehydrogenase;
GNRHR, gonadotropin releasing hormone receptor; FSHR, follicle
stimulating hormone receptor; LHR, luteinizing hormone receptor;
BCL-2, B-cell lymphoma-2.RT–PCR was carried out with an initial incubation at 95°C for
45 seconds, followed by 40 cycles of 95°C for 10 seconds and
annealing for 40 seconds; real-time fluorescence data were obtained
throughout this time. A melting-curve methodology was used to heat the
reactions from 60°C to 95°C in 0.5°C 15 s increments
while collecting fluorescence data to evaluate the specific amplification.
The relative abundances of the distinct mRNA molecules were estimated using
the 2−ΔΔCt technique [21] after all data were standardized to the internal
reference GAPDH. Each PCR result was tested for specificity using 1.8%
agarose gel electrophoresis followed by sequencing.
Calculation and statistical analysis
The results for litter size (total and alive), litter weight, and individual
weight are presented as the means and standard errors. Data were compared using
one-way repeated analyses of variance (ANOVAs, SPSS software version 22.0) with
Bonferroni post hoc tests. Simultaneously, nonparametric chi-square testing was
used to analyse conception and kindling rates, as well as preweaning mortality
[22]. A statistically significant
p value of 0.05 was considered.In the serial sampling experiment, relative mRNA abundances were calculated using
one-way ANOVA testing for the four monochromatic light hues’ major
treatments. The mean differences for each therapy were compared using the mean
standard error of the mean, and P 0.05 was judged significant. The means were
ranked using Duncan’s multiple range test. IBM SPSS software was used for
statistical analysis (ver. 13.0, IBM SPSS, Armonk, NY, USA).
RESULTS
Influences of light colour on nulliparous rabbit reproductive parameters,
litter size and kit weight during lactation
As shown in Table 2, red light and white
light affected the conception rate and kindling rate and increased the total
litter size at birth (p < 0.05). The effects of red
light on litter size at weaning, litter weight at weaning, and individual weight
at weaning increased compared with the green and blue groups. The effects of red
light on live litter size at birth were increased compared with those in the
blue group (p < 0.05).
Table 2.
Influences of light colour on rabbit does reproductive parameters,
litter size and kit weight during lactation
Item
White (control)
Red
Green
Blue
No. of does subjected to AI
266
268
265
269
Conception rate (%)
76.6
88.6
70.9
62.0
No. of kindling does
186
213
123
121
Kindling rate (%)
69.9
79.4
46.4
44.9
Total litter size at birth (n)
8.59±0.24[a]
8.72±0.13[a]
8.07±0.27[b]
8.01±0.39[b]
Live litter size at birth (n)
7.84±0.26[ab]
8.01±0.26[a]
7.75±0.32[ab]
7.50±0.41[b]
Litter weight at birth (g)
625.4±14.0[a]
623.4±11.0[a]
617.4±18.3[a]
622.5±28.0[a]
Litter size at weaning (n)
6.43±0.07[ab]
6.77±0.08[a]
6.19±0.07[b]
6.01±0.10[b]
Litter weight at weaning (kg)
6.51±0.09[ab]
7.43±0.07[a]
5.92±0.11[b]
5.54±0.17[b]
Individual weight at weaning (g)
1,013±6[ab]
1,098±9[a]
956±13[b]
922±11[b]
Preweaning mortality (%)
17.98
15.48
20.12
19.86
Different letters in the same row denote a difference
(p < 0.05).
No marked letter above the bar indicates that the difference is not
significant.
AI, artificial insemination.
Different letters in the same row denote a difference
(p < 0.05).No marked letter above the bar indicates that the difference is not
significant.AI, artificial insemination.
Numbers of follicles of different stages in female rabbits exposed to
different monochromatic light colours
Primitive follicles are located outside the cortex in great numbers. As shown in
Fig. 3, compared to white light, green
and blue light reduced the number of secondary follicles (p
< 0.05). Compared to red light, green and blue light reduced the number
of tertiary follicles (p < 0.05).
Fig. 3.
Numbers of follicles of different stages in female rabbits exposed to
different monochromatic light colours.
The values are presented as the means ± SEMs;
a,bDifferent letters on bars denote a difference
(p < 0.05). No marked letter above the bar
indicates that the difference is not significant.
Numbers of follicles of different stages in female rabbits exposed to
different monochromatic light colours.
The values are presented as the means ± SEMs;
a,bDifferent letters on bars denote a difference
(p < 0.05). No marked letter above the bar
indicates that the difference is not significant.
Relative abundance of GNRHR, FSHR, LHR, BCL-2, BAX, and FOXO1 mRNA in the
ovaries of female rabbits exposed to different monochromatic light
colours
As shown in Fig. 4, compared with white
light, red LED light resulted in greater ovarian FSHR and LHR mRNA expression
(p < 0.05). Compared with green and blue LED light,
red LED light resulted in greater BCL-2 mRNA expression (p
< 0.05). Compared with green LED light, red LED light inhibited FOXO1
mRNA expression in rabbit ovaries (p < 0.05).
Fig. 4.
Relative abundance of GNRHR, FSHR, LHR, BCL-2, BAX, and FOXO1 mRNA in
the ovaries of female rabbits exposed to different monochromatic
light colours.
a,bDifferent letters on bars denote a difference
(p < 0.05). No marked letter above the bar
indicates that the difference is not significant. GNRHR, gonadotropin
releasing hormone receptor; FSHR, follicle stimulating hormone receptor;
LHR, luteinizing hormone receptor; BCL-2, B-cell lymphoma-2.
Relative abundance of GNRHR, FSHR, LHR, BCL-2, BAX, and FOXO1 mRNA in
the ovaries of female rabbits exposed to different monochromatic
light colours.
a,bDifferent letters on bars denote a difference
(p < 0.05). No marked letter above the bar
indicates that the difference is not significant. GNRHR, gonadotropin
releasing hormone receptor; FSHR, follicle stimulating hormone receptor;
LHR, luteinizing hormone receptor; BCL-2, B-cell lymphoma-2.
DISCUSSION
In comparison to studies on the effects of light colour on avian reproductive
performance, there have been comparatively few publications on the effects of LED
light colour on rabbit reproductive performance. Existing research has tended to
focus on the effect of photoperiod on rabbit reproductive success. Sendrő et
al. [23] discovered that switching from an 8
h light period to a 16 h light period 8 days before insemination on a large-scale
rabbit farm effectively boosted the oestrus rate of female rabbits. According to
Sendrő Z et al. [23], compared to
normal white light, blue light has a positive influence on litter weight at 23 days
of age. The influence of light colours is mostly due to differences in light
wavelengths. Wu et al. [22] found that light
colour had no discernible effect on the conception and kindling rates of female
rabbits, but in this large sample study, we discovered that red and white light
affected the conception and kindling rates and increased the number of kits at birth
(total litter size and live litter size) (p < 0.05, Table 2). The effects of red light on litter
size and litter weight at weaning were superior to those of other light colours
(p < 0.05, Table
2). Because of its long wavelength, red light has the greatest biological
permeability [14]. It has been utilized in
photobiomodulation treatment to stimulate brain function [24]. It is thought that red light may infiltrate the cerebral
brain of rabbits and produce biological responses, ultimately improving rabbit
health. Based on our findings, we advocate replacing other colours of light with red
light in rabbit farms.GNRHR, FSHR, and LHR are significant genes associated with mammalian reproductive
success and play key roles in follicle growth and maturation, as demonstrated by
Zerani et al. [12] and Ramakrishnappa et al.
[25]. Few studies have been conducted on
the effects of different light colours on rabbit reproductive performance, and
current studies have tended to focus on the effects of photoperiod and light
intensity on female rabbit reproduction and associated genes. Sun et al. [26] investigated the influence of light
intensity on ovary gene expression, reproductive success, and body weight in rabbit
does with three different light intensities: 60 lx, 80 lx, and 100 lx. The relative
abundance of growth hormone receptor (GHR) mRNA expression was more abundant in 60
lx than in 80 lx or 100 lx (p < 0.05); at first
insemination, second insemination, and the second postpartum period
(p < 0.05), the bodyweight of the dose in Group 60 lx
was higher than that in the other two groups. For the first time, the findings of
this study show that different LED light colours can influence the expression of
FSHR and LHR mRNA in female rabbits (as shown in Table 2). Although rabbits are nocturnal creatures and hence less
sensitive to light colour than animals that are active during the day, substantial
differences in several features were discovered between groups lit by white, red,
green, and blue light. According to our findings, red light can influence the
reproductive success of inulliparous doesrabbits as well as the expression of
essential genes for follicular development. When compared to white light, red LED
light led to higher ovarian FSHR and LHR mRNA expression (p
< 0.05). Red LED light increased BCL-2 mRNA expression (p
< 0.05) compared to green and blue LED light. Red LED light decreased FOXO1
mRNA expression in female rabbit ovaries (p < 0.05) when
compared to green LED light. As a result, the prospective influence of light colour
merits further investigation.Numerous studies on the influence of environmental variables on the development of
female mammalian follicles have been performed, and some of them have achieved
significant progress [17]. For example, Shen
et al. [27] discovered that oxidative stress
generated by environmental variables affects follicle growth. FOXO transcription
factors are recognized as important mediators of oxidative stress and apoptotic
regulation [28]. FOXO1 was found to be
involved in oxidative stress-induced apoptosis of mouse follicular GCs both
in vivo and in vitro in mice. When mice were
treated with the oxidant, it was shown that higher apoptotic signals were associated
with increased expression of FoxO1 in GCs [29]. FOXO1 expression was also upregulated in proapoptotic and antioxidative
genes [27,28,30]. Red light substantially
decreased the expression of FOXO1 mRNA in this research. This finding is consistent
with the data on tertiary follicle number given in Fig. 2. Red light has a longer wavelength than other visible light
colours, and it contains less energy than other colours of light. The oxidative
stress imparted to the retina may have been milder in the red light group than in
the other groups, resulting in considerably lower FOXO1 mRNA transcription levels in
the red light group than in the other groups. It is hypothesized that the oxidative
stress response in female rabbits differs following different LED light treatments.
These variations may be due to the varied photon energy conveyed by different
wavelengths of LED light. When varied energy levels of LED light contact the retina,
the biological efficiency changes, resulting in variances in FOXO1 mRNA expression
levels in female rabbit ovaries.Throughout the oestrus cycle, follicular development is linked to granulosa cell
death, as explained in the introduction. The BCL-2 gene family is primarily involved
in granulosa cell apoptosis, and BCL-2 and BAX are important apoptotic genes. BCL-2
can prevent additional apoptosis by blocking the final route of apoptosis, whereas
BAX has the opposite effect and can accelerate apoptosis [14]. In this work, we chose two genes with distinct roles and
examined their expression patterns in female rabbits exposed to various LED light
colours. The results revealed that red LED light resulted in greater BCL-2 mRNA
expression (p < 0.05), we did not clarify the exact process,
and will investigate it more in the future work.
CONCLUSION
Although rabbits are nocturnal creatures and hence not as sensitive to light colour
as animals that are active during the day, there were substantial differences in
several features between groups lighted by white, red, green, and blue light. Red
light has been shown to have a positively effect on the reproductive success of
rabbits as well as the expression of critical genes for follicular development.Furthermore, according to common sense, LED lamps are more energy efficient than
incandescent lamps, it is recommended to promote the use of LED lights in the
production of domestic rabbits to reduce energy consumption.
Authors: Dorela D Shuboni; Shannon L Cramm; Lily Yan; Chidambaram Ramanathan; Breyanna L Cavanaugh; Antonio A Nunez; Laura Smale Journal: Physiol Behav Date: 2014-10-28
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.