DOI: 10.1111/jpn.

12382

ORIGINAL ARTICLE

Dynamics of anterior pituitary immunoreactive gonadotrophs in

moulted hens supplemented with protein, symbiotic and
probiotics
H. Anwar1 and Z. U. Rahman2
1 Department of Physiology, Government College University, Faisalabad, Pakistan, and
2 Institute of Pharmacy, Physiology and Pharmacology, University of Agriculture, Faisalabad, Pakistan

Summary
The present work delineates redistribution patterns of the hormone-producing cells of the anterior pituitary, after
the phase of moulting. Two hundred single comb White Leghorn hens at the end of their first production cycle
(Age = 70 week) were purchased from the commercial poultry farm and were induced to moult by high-dietary
zinc (3 g/kg feed/day) after 1 week of acclimatization, at the experimental research station, Department of Physi-
ology and Pharmacology, University of Agriculture, Faisalabad. The moulted birds were equally (n = 50) and
randomly allocated to their respective groups as G1 (control; CP (Crude protein) 16%, no supplement), G2
(CP18%, no other supplement), G3 (CP16%, symbiotic at does rate of 85 mg/l in drinking water daily) and G4
(CP16%, probiotic at dose rate of 85 mg/l in drinking water daily). Ten birds were slaughtered in each group at
5% and at peak of post-moult production stage to collect their pituitary glands. An earlier post-moult production
recovery, sustained and lengthier production span was seen in the G2 as compared to all other groups. The low-
est production and an earlier production decline were seen in G1. The cell diameter and area of follicle-stimulat-
ing hormone (FSH) and luteinizing hormone (LH) gonadotroph increased (p ≤ 0.01) in G2 and G3 as compared
to G1. The FSH gonadotroph nucleus diameter and area did increase (p ≤ 0.01) in G2 and G3, while LH gonado-
troph nucleus diameter and area decreased (p ≤ 0.01) in G2 and G3 as compared to G1. The increased FSH and
LH gonadotroph diameter in protein and symbiotic supplemented birds is accountable for the increased egg pro-
duction in these groups.
Keywords gonadotrophs, immunohistochemistry, protein, probiotic, moulted layers

Correspondence H. Anwar, Incharge, Department of Physiology, Government College University, Allama Iqbal Road, Faisalabad-38000, Pakistan.
Tel: +92-41-9200760; Fax: +92-41-9201419;; E-mail: drhaseebanwar@gcuf.edu.pk

Received: 22 April 2014; accepted: 29 June 2015

Introduction
Induced moulting of laying hens at the end of their
production cycle is economically beneficial as com-
pared to culling of the birds and rearing new chicks
after the completion of first production cycle. How-
ever, after escalating controversies about the harms
of fast induced moulting and demand for the ban of
induced moulting, alternate induced moulting
methods along with supplementation programs of
moulted layers are being investigated. High-dietary
zinc-induced moulting proves to be a increased
moulting method (Sandhu et al., 2007) along with
protein and probiotic supplementation (Anwar et al.,
2012b). The redistribution of the hormone-produc-
ing cells of the anterior pituitary was observed in
the birds after the phase of moulting (Sandhu et al.,
2010).
FSH and LH gonadotrophs are among the five major
hormones released from the anterior pituitary (adeno-
hypophysis) of chicken. The anterior pituitary can be
demarcated into cephalic and caudal lobes in avian
species, while the intermediate lobe is not present.
Hormone-producing cells are scattered throughout
the cephalic and caudal lobes (Puebla-Osorio et al.,
2002), while intermediary lobe is not present in the
birds (Ramesh et al., 1998). The enhanced release of
LH was reported in the plasma due to increased
responsiveness of pituitary in the release of LHRH in
the post-moulted hens (Sharp et al., 1992). Another
study revealed that the LH concentration in the
plasma was decreased just after moulting and replen-
ished after re-feeding for a week in the hens (Tanabe
et al., 1981). During induced moulting, circulating
levels of luteinizing hormone (LH), oestrogen and
progesterone decrease (Dickerman and Bahr, 1989).

448 Journal of Animal Physiology and Animal Nutrition 100 (2016) 448–455 © 2015 Blackwell Verlag GmbH
H. Anwar and Z. U. Rahman
Birds on the comparatively high protein diet (18%)
in their post-moult phase recovered earlier from the
moulting stress (Anwar et al., 2012b). The protein
and probiotic supplementation in the diet of the post-
moult birds have shown the impact on increasing the
diameter of growth hormone-producing cells and
decreasing the diameter of lactotrophs (Anwar et al.,
2012a). Similarly, Haddadin et al. (1996) reported
that egg production and egg quality also improved by
supplementing the basal diet with liquid culture of
Lactobacillus acidophillus. However, the reported litera-
ture is lacking regarding the impact of protein and
probiotic supplementation on the dynamics of pitu-
itary gonadotrophs in moulted layers with relation to
the post-moult production performance. Therefore,
this study was designed in this context.
Materials and methods
The 70 weeks old commercial single comb spent
White Leghorn layers (n = 200) were purchased from
a commercial poultry farm and brought to the poultry
research station of the Department of Physiology and
Pharmacology, University of Agriculture, Faisalabad.
After 1 week of acclimatization, the birds were
induced to moult by inclusion of dietary ZnO in a con-
centration of 3 g/kg of the feed (Table 1). The speci-
fied groups and their treatments were allotted as
group G1 (Control, CP 16%, No supplement), G2 (2%
Protein supplement; CP 18%, No other supplement),
G3 [CP 16%; Symbiotic (Perfectinâ Diasham
Resources, Singapore city, Singapore) in water at a
dose level of 85 mg/L of drinking water daily) and G4
(CP 16%; Probiotic (Protexinâ Probiotics UK Interna-
tional Marketed by Hilton Pharma, Karachi, Pakistan)
at a dose level of 85 mg/L of drinking water daily after
the completion of moulting process of 4 week (50
Table 1 Moulting schedule of spent White Leghorn hens through
high-dietary zinc at a dose level of 3 g/kg feed
Feed offered Age
Stage (g/bird/day) (Weeks)
Pre-moult acclimatization 110 70
phase*
Phase of moulting† 35 71–74
Rest period‡ 70 74–75
80 76
Post-moult production 110 77–90
phase
*Deworming, EDS and ND vaccination.
†Respective treatments of all the groups were started.
‡Start of egg production.
Journal of Animal Physiology and Animal Nutrition 100 (2016) 448–455 © 2015 Blackwell Verlag GmbH
Morphometry of gonadotrophs in hens
birds in each group). The composition of feed and sup-
plements has been presented in Table 2. Twenty birds
were sacrificed at peak of second production cycle to
collect their pituitary glands. The temperature of the
experimental shed was maintained at 25  2 °C
throughout the experiment period. The egg produc-
tion record was maintained throughout the experi-
mental period in each group. All the experimental
procedures were adopted after the approval from
Institutional ethical review committee for animal care
of University of Agriculture, Faisalabad, Pakistan.
Collection, preservation and sectioning of pituitary
glands
After sacrificing (decapitation) the birds, pituitary
glands were collected as follows. The skull bone was
made accessible by reflecting over the skin and a cup-
shaped cut was given on the upper part of the skull.
The brain was removed carefully from the skull in
intact form without breaking it from its base. Pituitary
gland was identified right at the base of the brain in a
fossae-like depression just beneath the hypothalamus.
Pituitary glands were removed carefully and initially
kept in Bouin’s Hollande solution for 24 h then they
were shifted to 4% formalin solution and preserved
till the paraffin blocking. Before the paraffin blocking,
pituitary glands were dehydrated through a series of
alcohol. Microtome (Microm, HM 315, Ramsey, MN,
USA) was used to cut 4 lM thick pituitary sections.
The cut sections were floated in a water bath adjusted
at 60 °C. The appropriate sections were selected and
lifted on the poly-L-lysine-coated cleaned (1% HCl in
70% alcohol) slides (Culling et al., 1998). Before the
start of immunostaining, selected sections were rehy-
drated by standard rehydration procedure. The rabbit
raised antisera (primary antibodies) against ovine beta
FSH and ovine beta LH were provided by the courtesy
of Dr. A. F. Parlow (National Hormone and Peptide
Program, Harbor-UCLA Medical Center, 1000 W; Car-
son St., Torrance, CA, USA).
Light
(Hours) Water
Localization of Immunoreactive gonadotrophs
16 Ad-lib
Rabbit-specific secondary antibody (Biotinylated, goat
12 Ad-lib antirabbit IgG; H+L) HRP/DAB detection immunohisto-
12 Ad-lib chemistry kit was provided by Abcam (Cat # ab64261),
14 Ad-lib Abcam plc, Cambridge, UK. The Aqua hold barrier
16 Ad-lib Pap pen (ProSciTech, PST, Thuringowa QKl, 4817,
Australia) was used encircle the sections to hold the
drops of reagents and antibodies over the sections and
avoiding their out flow. The slides were removed from
PBS to start the staining procedure. Before to start, the
449
Morphometry of gonadotrophs in hens H. Anwar and Z. U. Rahman

Table 2 Composition of supplements and feed (g/100 gm) for moulted White Leghorn hen

CP 16% CP 18% Composition of symbiotic Composition of probiotic

energy = 2795 Kcal energy = 2800 Kcal (Perfectin) (Per Kg) (Protexin)
Feed ingredients g/100 g g/100 g probiotic: viability: 9 104 cfu/ml viability: 1 9 106 cfu/ml

Corn 40 40 Lactobacillus acidophilus

Rice tips 10 10 Bifidobacterium thermophilus Lactobacillus plantarum
Rice polishing 11 11 Bifidobacterium longum
Maize gluten 30% 6 – Streptococcus faecium Lactobacillus bulgaricus
Maize gluten 60% – 4 Prebiotics
Canola meal 10 10 VitA 4 000 000 IU Lactobacillus acidophilus
Soya bean meal 8 10 Vit. D3 800 000 IU
Fish meal 6 6 Vit. E 500 IU Lactobacillus rhamosus
Dicalcium phosphate 1.5 1.5 Vit. K 200 mg
Limestone powder/0 Chips 7 7 Vit. B1 200 mg Bifidobacterium bifidum
Vitamin premix* + amino acid† 0.5 0.5 Vit B2 2000 mg
Total 100 100 Vit B6 600 mg Streptococcus thermophilus
Vit C 2000 mg
Folic acid 100 mg Enterococcus faecium
Niacin 10 000 mg
L-lysine 5000 mg Aspergillus oryzae
Dl-methionine 15 000 mg
Iron 7500 mg Candida pintolopesi
Copper 1000 mg
Zinc 7500 mg
Manganese 10 000 mg

Study Groups: G1 – (CP16%, no other supplement), G2 – (CP18%, no other supplement), G3 – (CP16%, Symbiotic at dose rate of 1 g/4 l drinking water),
G4 – (CP16%, Probiotic at dose rate of 1 g/4 l drinking water).
*Composition per kg of diet: vitamin A, 8300 IU; cholecalciferol, 2200 ICU; vitamin E, 8 IU; vitamin B12, 0.02 mg; riboflavin, 5.5 mg; D-calcium pan-
tothenic acid, 15 mg; niacin, 36 mg; choline, 500 mg; folic acid, 0.5 mg; vitamin B1, 1 mg; pyridoxine, 2.2 mg; biotin, 0.05 mg; vitamin K, 2 mg.
†Composition per Kg of diet: methionine 0.143 g, lysine 0.72 g, threonine 0.35 g.

immunostaining hydrogen peroxide block (Abcam,
ab64261) was applied to the section for 10 min at
room temperature to stop the activity of endogenous
peroxidases. Four washings of PBS were given
followed by a drop of protein block (Abcam, ab64261)
over the section for 5 min. A single PBS washing was
given and the working dilutions of obFSH (1:100) and
obLH (1:100) primary antibody were made in PBS and
applied to different sections and marked for specific
cell. Two hours of incubation at room temperature
were given with primary antibody. The tissue sections
were washed four times with buffer after the
completion of appropriate incubation. Ten minutes of
incubation with rabbit-specific secondary antibody
(Biotinylated goat antirabbit IgG) were given at room
temperature. Thereafter, four washings with buffer
were given. Then, a drop of enzyme streptavidin
peroxidase was poured over the tissue and incubated
at room temperature for 10 min. Four buffer rinses
were given thereafter. The chromogen dilution was
made with DAB substrate (1:50) freshly, vortexed to
mix and applied to the tissue for 10 min at room tem-
perature. Five washings to DAB chromogen were
given with buffer. Before covering with the cover slip,
the slide was air-dried and mounted with DPX moun-
tant.
Specificity of immuno reactive cells (negative control)
The primary antibody was removed or replaced with
PBS/1% normal rabbit serum, or secondary antibody
was raised in inappropriate specie (other than rabbit)
in negative control slides (Photomicrograph 1 Pitu-
itary gland immunoreactive FSH-gonadotrophs in
moulted White Leg-Horn.). The absence of immune
reaction in these slides confirms the specificity of
immunoreactive cells (Anwar et al., 2012a).
Morphometry of immuno reactive gonadotrophs
One most appropriate slide from each pituitary tissue
was selected, and then, calculations were made as fol-
lows. The tissue sections were located on the slides
and observed under microscope. The specified
immunoreactive FSH and LH gonadotrophs were
identified. More than 300 immunoreactive gonado-
trophs in three sections on each slide (Total number ≥
6000) were observed for average morphometric calcu-

450 Journal of Animal Physiology and Animal Nutrition 100 (2016) 448–455 © 2015 Blackwell Verlag GmbH
H. Anwar and Z. U. Rahman
lations. IMAGE J software (Image J 1.44P; Wayne Ras-
band, National Institute of Health, Bethesda, MD,
USA) was used for the computerized morphometric
calculation of the cell and nucleus diameter (lm) and
area (lm2) for each immunoreactive gonadotroph
(FSH & LH).
Statistical analysis
The analysis of variance technique was applied in
non-factorial completely randomized experimental
design. The significance between the means of
treatment groups and their standard error was deter-
mined. Duncan multiple range test was applied to
Journal of Animal Physiology and Animal Nutrition 100 (2016) 448–455 © 2015 Blackwell Verlag GmbH
Morphometry of gonadotrophs in hens
rank the significant means between groups. p Value
was considered significant at p < 0.05.
Results
Egg production performance
The post-moult egg production in different groups (al-
most 20 weeks long) is presented in Fig. 1. Almost
after 1 week, 5% egg production was observed in
moulted hens from G2 and G3 group as compared to
control (G1) in which the 5% production was seen
after 2 weeks. An increased egg production was
observed in G2 as compared to all other groups
throughout the peak production phase. The decline of
production at the end of production was abrupt in G1;
however, a delayed decline in production was seen in
G2, G3 and G4. Overall in average egg production,
16% in G2 and 10% in G3 and G4 increased egg pro-
duction was seen as compared to the G1 throughout
the experimental period (Fig. 2).
Morphometry of immunoreactive gonadotrophs
A significant (p ≤ 0.01) increase of the overall mean
cell diameter in G2 and G3 group was seen as com-
pared to G1 and G4 (Fig. 3); however, the overall
mean cell area did increase (p ≤ 0.01) in G2 and G3 as
compared to G1, but between G3 and G4, there was
no significant difference (Fig. 3). The FSH gonado-
troph nucleus diameter increased significantly in G2
and G3 as compared to G1 and G4; however, even big-
ger (p ≤ 0.01) nucleus diameter was seen in G2 as
compared to G3 (Fig. 4). A similar trend was observed
in the nucleus area as it increased (p ≤ 0.01) in G2
and G3 as compared to G1 (Fig. 4). However, the
overall mean nucleus area did not differ significantly
between G3 and G4 groups. The immunoreactive FSH
gonadotrophs in different groups have been presented
in the photomicrograph 2.
The significant increase (p ≤ 0.01) in the overall
mean LH gonadotroph diameter as well as the area
was observed in G2 and G3 as compared to G1
(Fig. 5). However, the cell diameter and cell area did
not differ (p ≥ 0.01) between G3 and G4. The overall
mean LH gonadotroph nucleus diameter was low
(p ≤ 0.01) in G2 and G3 as compared to G1 and G4
which did not differ from each other (Fig. 6). Like-
wise, overall mean nucleus area did decrease signifi-
cantly in G2 and G3 as compared to G1 and G4
(Fig. 6). The immunoreactive LH gonadotrophs have
been presented in photomicrograph 3. Negative Control
slides have been presented in photomicrograph 1.
451
Morphometry of gonadotrophs in hens H. Anwar and Z. U. Rahman

120 G1
G2

100 G3
G4

80

60

40

20

Fig. 1 Post-moult egg production (start at

0 77-week age) percentage in control (G1), 18%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
protein (G2), symbiotic (G3) and probiotic (G4)
Post molt prodcution (Weeks) supplemented groups.

80
Discussion
70
The increased cell diameter and area of FSH gonado-
60 troph in G2 and G3 is attributed to the early resump-
G2 tion of laying as well as the enhanced egg production
G3 G4
50
Production %

G1 74.51 64.95 65.03 performance in these groups. It was reported that in

58.51
40 the birds showing the early resumption of egg produc-
tion after a phase of fast-induced moult (Chowdhury
30
and Yoshimura, 2002), not only the number of FSH
20 gonadotrophs increased, their cell diameter also
increased which could be responsible for this early
10
resumption of egg laying. The increased nucleus diam-
0 eter and area in FSH gonadotrophs indicates the
hyperactive nucleus, which may be due to the
Fig. 2 Average egg production % age in control (G1), 18% protein (G2), increased mitosis and proliferation of the cells. The
symbiotic (G3) and probiotic (G4) supplemented groups throughout
increased diameter and area of these gonadotrophs
post-moult production phase (20 week).
(FSH and LH) could be linked with the decrease in the
diameter of lactotrophs. The cell and nucleus diameter
Localization of immunoreactive gonadotrophs
as well as the area of lactotrophs decreased in the pro-
Immune reactive LH and FSH were found equally tein, symbiotic and probiotic supplemented moulted
distributed in both cephalic and caudal lobes in all the layers, which was previously reported by the authors
groups without any significance between groups. Sig- (Anwar et al., 2012a). The early recovery of produc-
nificantly increase number of spindle-shaped onto- tion in symbiotic and probiotic supplemented moulted
genic LH as well as FSH cells were found in the birds after the phase of moulting assumed to be
control groups as compared to the other groups. related to the decreased lactotroph as well as the

30
A 700
A A
25 B 600
Cell diameter (µm)

B B
500
Cell area (µm2)

20
BC
400 Fig. 3 Overall mean FSH gonadotroph
15 C
diameter (mean  SE) and area (mean  SE) in
300
10 control (G1), 18% protein (G2), symbiotic (G3)
200 and probiotic (G4) supplemented groups
5
100 (N = 25 each) at peak of post-moult production
0 0 (11th week). A–CSimilar alphabets on the bars
G1 G2 G3 G4 G1 G2 G3 G4 do not differ significantly.

452 Journal of Animal Physiology and Animal Nutrition 100 (2016) 448–455 © 2015 Blackwell Verlag GmbH
H. Anwar and Z. U. Rahman Morphometry of gonadotrophs in hens

16 180
A A
14 160

Nucleus diameter (µm)

12 140

Nucleus area (µm2)

120
10 B
C 100
Fig. 4 Overall mean FSH gonadotroph nucleus 8
C 80 B
diameter (mean  SE) and area (mean  SE) in 6
control (G1), 18% protein (G2), symbiotic (G3) 60 BC
C
4
and probiotic (G4) supplemented groups 40
(N = 25 each) at peak of post-moult production 2 20
(11th week). A–CSimilar alphabets on the bars 0 0
do not differ significantly. G1 G2 G3 G4 G1 G2 G3 G4

increased gonadotrophs diameter. Previously, it was
reported by Kirby et al. (1979), which hyperpro-
lactinemia causes the malfunctioning of the hypotha-
lamic–pituitary axis, gonads and the adrenal cortex.
This leads to the increased nesting behaviour and
stress hormone (cortisol) level, which ultimately
responsible for the decreased production. The reduc-
tion in the serum cortisol concentration in the protein,
probiotic and synbiotic supplemented moulted laying
hens strengthens this verdict (Anwar, 2011). More-
over, decreased lactotroph diameter in current experi-
ment indicates the decrease in prolactin level, which
is an indirect indication of overall better egg produc-
tion in the supplemented birds. It was reported that
development of hyperprolactinemia due to the decline
of the egg production also decreases the LH respon-
siveness, which further reduces the egg production
(Ramesh et al., 1996). The LH is responsible for the
sexual maturity and egg production; it is also involved
in the ovulation and oviposition with other sex ster-
oids including progesterone (Puebla-Osorio et al.,
2002). FSH and LH gonadotrophs were distributed
throughout the cephalic and caudal lobe in this study,
which has also been reported in some previous studies
(Berghman et al., 1993) but in past, some researchers
have also reported their restricted localization in the
caudal lobe (Wada and Asai, 1976). The increased
localization of irLH at the peripheries of the anterior
pituitary in G2 and G3 has also been reported (Proud-
man et al., 1999). The presence of spindle-shaped
irLH cells in G1 and their major localization at the
margins of the anterior pituitary in zinc-induced,

Journal of Animal Physiology and Animal Nutrition 100 (2016) 448–455 © 2015 Blackwell Verlag GmbH 453
Morphometry of gonadotrophs in hens H. Anwar and Z. U. Rahman

35
A 900
30 AB
800 A
Cell diameter (µm)

BC
25 C 700 B

Cell area (µm2)

600 BC
20 Fig. 5 Overall mean LH gonadotroph diameter
500 C
15 400 (mean  SE) and area (mean  SE) in control
10 300 (G1), 18% protein (G2), symbiotic (G3) and
200 probiotic (G4) supplemented groups (N = 25
5
100 each) at peak of post-moult production (11th
0 0 week). A–CSimilar alphabets on the bars do not
G1 G2 G3 G4 G1 G2 G3 G4 differ significantly.

10 70
A
9 A
A
8 60 A
Nucleus diameter (µm)

B Nucleus area (µm2)

B
7 50 B
6 B
40
5 Fig. 6 Overall mean LH gonadotroph nucleus
4 30 diameter (mean  SE) and area (mean  SE) in
3 20 control (G1), 18% protein (G2), Ssmbiotic (G3)
2 and probiotic (G4) supplemented groups
10
1 (N = 25 each) at peak of post-moult production
0 0 (11th week). ABSimilar alphabets on the bars do
G1 G2 G3 G4 G1 G2 G3 G4 not differ significantly.

moulted, White Leghorn layers has also been reported protein, symbiotic and probiotic did help to reduce the
by Nakamura et al. (2004) and Sandhu et al. (2008). oxidative stress by improving the antioxidant status of
The impact of under nutrition on the pituitary the body (Anwar et al., 2012b).
response in the animals is multidimensional as Her- The supplementation of the moulted birds with
bert (1980) observed a decrease in LH-releasing hor- additional protein and symbiotic did show an impact
mone from the hypothalamus in low-protein fed on the earlier post-moult production recovery, an
animals. The supplementation of moulted birds with increased production potential with a delayed decline

454 Journal of Animal Physiology and Animal Nutrition 100 (2016) 448–455 © 2015 Blackwell Verlag GmbH
H. Anwar and Z. U. Rahman Morphometry of gonadotrophs in hens

in the production (Fig. 2). The increased production
performance is connected with improved cellular
dynamics of gonadotrophs in supplemented groups.
Among supplementation, additional protein supple-
mentation did show the most promising results
regarding production performance and morphometry
of anterior pituitary gonadotrophs.
The rejuvenation of the hormone-producing cells of
the pituitary is evident in this study, and supplemen-
tation did show the increased FSH and LH gonado-
troph diameter in protein and symbiotic
supplemented birds. This increase in the diameter is
responsible for the increased production of these hor-
mones, which is ultimately accountable for the
improved egg production in these supplemented
groups.
Acknowledgements
The authors are thankful to the Higher Education Com-
mission, Islamabad, Pakistan for extending the grant
under PhD Indigenous scholarship scheme, Batch-III.
Conflict of interests
The authors declare no conflict of interests.

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