for review only - university of toronto t-space · for review only spermatogenesis in the guinea...

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Spermatogenesis and cellular associations in the

seminiferous epithelium of Guinea cock (Numida meleagris)

Journal: Canadian Journal of Animal Science

Manuscript ID CJAS-2016-0068.R1

Manuscript Type: Article

Date Submitted by the Author: 22-Jul-2016

Complete List of Authors: Abdul-Rahman, Ibn Iddriss; University for Development Studies, Animal

Science Obese, F.Y.; University of Ghana, Animal Science Robinson, J. E.; University of Glasgow

Keywords: Gamete, Poultry, Reproductive physiology, Semen, Sperm physiology

https://mc.manuscriptcentral.com/cjas-pubs

Canadian Journal of Animal Science

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SPERMATOGENESIS IN THE GUINEA FOWL

1

Spermatogenesis and cellular associations in the seminiferous epithelium of

Guinea cock (Numida meleagris)

Abdul-Rahman Iddriss Ibn 1, †, Obese Frederick Yeboah

2, and Robinson Jane

3

1Department of Animal Science, Faculty of Agriculture, University for Development Studies, P. O.

Box TL 1882, Nyankpala Campus, Tamale, Ghana,

2Department of Animal Science, School of Agriculture, University of Ghana, P. O. Box LG 226,

Legon, Ghana,

3Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical,

Veterinary and Life Sciences, University of Glasgow, Bearsden Road, Glasgow G61 1QH,

Scotland, UK.

ABSTRACT

The study describes the steps of spermiogenesis and stages of seminiferous epithelial cycle in

breeding guinea cocks. Epoxy resin embedded sections were employed for the determination of the

stages of seminiferous epithelial cycle in the guinea fowl. Acrosomic granules aided in identifying

the initial steps of spermiogenesis, while nuclear morphological changes facilitated the

identification of subsequent stages. Eleven steps of spermiogenesis and 9 stages of seminiferous

epithelium were recognised in the guinea fowl testis. Three spermatogonial types, namely,

spermatogonial A, B and intermediate types were identified in the guinea fowl seminiferous

epithelium. Spermatogonial A and preleptotene spermatocytes had the largest (p < 0.05) nuclei

diameters in the seminiferous epithelium, while round spermatids had the least. Within germ cells,

no significant (p > 0.05) differences were found among birds in spermatogonial A and intermediate

spermatogonial nuclei diameters. Spermatogonial B, pre-leptotene primary spermatocyte, type I

† Corresponding author (e-mail: [email protected]/[email protected]).

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spermatocyte (primary spermatocyte in prophase I) and round spermatid nuclei diameters were,

however, bigger (p < 0.05) in some birds than others. The classification of the seminiferous

epithelial cycle in the guinea fowl was similar to that described in other birds and mammals.

Key words: Guinea fowl, germ cell, spermiogenesis, nuclear diameter

INTRODUCTION

Spermatogenesis involves several physiological processes including spermatocytogenesis, meiosis,

and spermiogenesis (Johnson et al. 2000). The kinetics of spermatogenesis have been amply

described (Jones and Lin 1993; Aire 2007). Spermiogenesis is the transformation of spermatids into

sperm cells without further cell division, and this has been studied extensively in mammals, and

most of our knowledge of the developmental and transformational processes in the spermatid is

derived from these studies (Aire 2014). Spermiogenesis in guinea fowls entails 10 distinct

morphological steps (Aire et al. 1980). Compared to 8 - 10 steps in Gallus gallus (de Reviers 1971;

Gunawardana 1977; Tiba et al. 1993) and 12 steps in Coturnix coturnix japonica (Lin et al. 1990;

Lin and Jones 1993). Spermiogenesis entails the formation of an acrosome and an axoneme, loss of

cytoplasm, and replacement of nucleohistones with nucleoprotamine, which accompanies nuclei

condensation (Olivia and Mezquita 1986; Sprando and Russell 1988).

There have been some disagreements about the cycle and wave of spermatogenesis in birds

(Clermont 1958; Yamamoto et al. 1967; Aire et al. 1980). The main problem in studying the bird is

that each cellular association in the seminiferous epithelium occupies only a small area of the

seminiferous tubule, and so it is difficult to determine the composition of the associations, and

consequently, the cycle of the epithelium and the spatial arrangement of the stages of the cycle in

the seminiferous tubule (Kirby and Froman 2000). Each cell type of the cell association is

morphologically integrated with the others in its developmental processes (Clermont and Perey

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1957). Most commonly, the youngest generation of spermatids are used to classify cell associations

because they have easily recognisable morphological features. In mammalian studies, the acrosomic

system stained by PAS has been mostly used in identifying spermatid differentiation (Orsi and

Ferreira 1978). Aire et al. (1980), however, reported that PAS staining was not a very satisfactory

method of identifying acrosome differentiation in the guinea fowl since recognition of the acrosome

became difficult after step 4 of spermiogenesis. Similar observations were made in ducks (Clermont

1958) and fowls (de Reviers 1971; Gunawardana 1977). In the Japanese quail, however, thin

toluidine blue stained sections gave sufficient resolution to identify developing acrosome of

spermatids up to step 5 of spermiogenesis (Lin et al. (1990). The authors further indicated that

nuclear morphological changes were very useful in identifying the remaining steps of

spermiogenesis.

There is still uncertainty as to whether cellular associations are found in the seminiferous

epithelium of all birds. For instance, while Noirault et al. (2006) reported no definite pattern of

cellular associations in the seminiferous epithelium of turkeys and domestic fowl, Lin and co-

workers (1990) described these associations in the seminiferous epithelium of Japanese quail

relying on only a single bird. Their reports, however, had some disagreements with those of

Yamamoto et al. (1967) in the same species. Moreover, much more is known about

spermatogenesis in mammals than in birds (Thurston and Korn 2000). The present study, therefore,

seeks to elucidate the steps of spermiogenesis and stages of cellular associations in the seminiferous

epithelium of guinea cocks, considering the fact that there is only one report on this phenomenon in

these birds Aire et al. (1980).

MATERIALS AND METHODS

Experimental Site

The birds used for the study were raised at the Poultry Unit of the Department of Animal Science,

University for Development Studies, Nyanpkala, Tamale (Ghana). Nyanpkala lies on latitude 9°

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69'N and longitude 0° 83'W. Temperatures are generally high with minimum and maximum values

of 22 °C and 35 °C recorded in March and December, respectively (SARI 2008). Rainfall is

monomial with mean annual rainfall varying from 1,000-1,500 mm and peaks from August to

September, with a relatively long dry season extending from November to April. The area lies in

the Guinea Savannah zone, and has nearly equal amounts of light and darkness (12L: 12D)

throughout the year. Guinea fowls (Numida meleagris) in this location generally breed during the

rainy season (May to October), and are not fertile during the dry season (November to April)

(Awotwi, 1987).

Animals and Management

A total of 10 local guinea cocks (Numidae meleagris), of the pearl variety, were used for the study.

Birds were brooded for 6 weeks (Teye and Gyawu 2002), and then transferred to a deep litter house

until the end of the experiment. They were individually identified using tags placed through their

inner wings to prevent detection by other birds and thus avoid pecking. Keets were brooded at 35°C

from hatching until three weeks of age (WOA), and then at 32°C until six WOA (Teye and Gyawu

2002). Birds were then maintained at ambient temperatures of between 22°C and 35°C until the end

of the experiment. Feed and water were supplied ad libitum. Day old keets were fed ground maize

in flat feeders, followed by a starter (22% crude protein and 3,000 Kcal ME/kg diet) ration from day

2 until 6 WOA. This was followed by a grower ration (14% crude protein and 2,800 Kcal ME/kg

diet) from 6 WOA until 21 WOA and then a layer feed until the end of the experiment (17.5%

crude protein and 2,800 Kcal ME/kg diet).

Information on lighting requirements of the local guinea fowls from hatching are unavailable,

and those used for chicken, are usually employed. In this case, however, the “golden rule” to

follow in designing lighting programmes for pullets (Thiele 2009) was followed. All birds received

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24 h light from day old until one-WOA, and this was reduced to 16 h until birds were 3 weeks old.

These longer light periods during the first 3 weeks of life were to ensure maximum feed

consumption, enough to ensure maximum growth, initially. This was gradually reduced to a

minimum of 12 h by the 7th

WOA, marking the phase of constant light (Thiele 2009). Thereafter,

birds were maintained under natural photoperiods (12L: 12D) until the end of the study.

Experimental Procedure and Histological Preparation

Animals used in this experiment were cared for under guidelines comparable to those laid down by

the Canadian Council on Animal Care. Birds were sacrificed at 34 WOA by cervical dislocation,

during the breeding season (May). Their testes were completely freed from the adjoining ligaments

and fascia, and weighed. Using the procedure previously described by Lin and associates (1990),

Single seminiferous tubules were separated from the testicular parenchyma by flushing the testes

with 1% (v/v) glutaraldehyde in 0.1M phosphate buffer (PH 7.2). Testes were cut into 5 mm lengths

and fixed in 3% (v/v) glutaraldehyde in phosphate buffer overnight, with post fixation in 1%

osmium tetraoxide.

Testicular tissues were embedded in epoxy resin (Sigma-Aldrich, Germany), sectioned 2 µm

thick with an ultramicrotome (Leica EM UC6), using glass knives, and stained with 1% toluidine

blue and 0.5% borax in 30% ethanol. Sections were then studied under immersion oil (x100

magnification).

Cell identification and Stereological Analysis

Spermatogonia were largely distinguished based on heterochromatin appearance and distribution,

and nuclear characteristics (Aire et al. 1980; Lin et al. 1990). In all cases, the location, relative size,

shape and nuclear morphology of germ cells helped in cell identification. Identification of the

various stages of seminiferous epithelium was facilitated by the finding that adjacent stages are

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separated from one another if the seminiferous tubules were previously flushed with 0.1M

phosphate buffer before fixation with glutaraldehyde (Lin et al., 1990). Nuclear diameters of

testicular germ cells were obtained with previously calibrated calipers (this was calibrated using

graticule under immersion oil) under immersion oil, using sections from 10 males and counting 20

nuclei/cell type/male.

Statistical Analysis

Data were analysed using the SPSS software, version 20.0 (IBM Corp. 2011). Differences in

nuclear diameters between germ cell types were analysed using Kruskal-Wallis test and medians

separated using Mann-Whitney U test, since variances were not homogenous. Differences within

germ cell types between individual animals were analysed using one-way repeated measures

Anova, and means separated using the Sidak correction test. The data were tested for sphericity

using the Mauchly’s test. Data were presented as either mean ± standard error of mean (SEM) or

Median {Interquartile range (IQR)}. All comparisons were done at 5% level of significance.

RESULTS

Description of Germ Cell Types

Three different types of spermatogonia were seen in mature guinea fowl testes, distinguished based

on heterochromatin appearance and distribution, and nuclear diameter (Aire et al. 1980; Lin et al.

1990), and were categorized into spermatogonial A, B and intermediate types, following the

nomenclature of Yamamoto et al. (1967). All these spermatogonia were located on the basement

membrane. The type A spermatogonia had large, pale staining spherical nuclei, with centrally

located intensely stained chromatin particles, consisting of 2-4 large clumps, and a few small

clumps. They were the largest (p < 0.05) of all the spermatogonia, averaging 6.4 (6.2-7.0) µm. The

type B spermatogonia on the other hand were distinguished by the presence of clumps of chromatin

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adhering to the nuclear membrane, they were more densely stained than the spermatogonia A, and

had ovoid nuclei with diameter {3.7 (3.6-3.8) µm} significantly smaller (p < 0.05) than the other 2

types of spermatogonia. The intermediate form had round to ovoid nuclei with a diameter of 4.6

(4.3-4.9) µm. They had more darkly staining nuclei than both A and B types.

In the pre-leptotene stage, the primary spermatocytes had nuclear diameter {6.4 (6.0-6.8) µm}

similar to spermatogonia A, and these two germ cell types had the largest diameters in the

seminiferous epithelium. These were followed in descending order by type I spermatocyte (primary

spermatocytes in prophase I) {4.9 (4.6-5.3) µm}, intermediate spermatogonia and spermatogonial

B. At pre-leptotene stage, primary spermatocytes had chromatin consisting of randomly dispersed

narrow filaments. During leptotene, the nuclei consisted of aggregation of thin filamentous

chromatin eccentrically located. The zygotene cell nucleus had chromatin consisting of thickened

filaments distributed uniformly in an irregular manner, while the pachytene cell nuclei had

thickened and tortuous chromatin strands distributed throughout the nucleus. The rarely observed

type II spermatocytes (secondary spermatocytes) were smaller than all previously observed germ

cells. The nuclei were spherical and characterised by thick clumps of chromatin, some of which

were associated with the nuclear membrane. The round spermatids had the smallest (p < 0.05)

diameter {3.2 (3.0-3.5) µm} of all germ cell series, and were located just above or between bundles

of filiform shaped spermatids and in the tubular lumen. They had round nuclei and showed

scattered clumps of chromatin.

Within germ cell nuclear diameter variability among individual birds were also explored, and are

presented in table 1. In all cases, Mauchly’s test indicated that the assumption of sphericity had

been violated, {(Spermatogonial A - X2 (5) = 340.67, p < 0.001, and therefore degrees of freedom

(df) were corrected using Greenhouse-Geisser estimates of sphericity (ε = 0.383)), (Spermatogonial

B - X2 (5) = 197.64, p < 0.001, and df corrected using ε = 0.514), (Intermediate spermatogonial - X

2

(5) = 228.90, p < 0.001, and df corrected using ε = 0.380), (Round Spermatid - X2 (5) = 99.84, p <

0.001, and df corrected using ε = 0.526), (Pre-leptotene primary spermatocyte - X2

(5) = 112.34, p <

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0.001, and df corrected using ε = 0.443), (Primary spermatocyte in prophase I - X2

(5) = 113.51, p <

0.001, and df corrected using ε = 0.533)}. No significant (p > 0.05) differences were found among

birds in spermatogonial A and intermediate spermatogonial nuclei diameters. Bird numbers 06 and

07, however, had significantly bigger (p < 0.05) spermatogonial B than bird number 02. Bird

number 06 had a significantly (p < 0.05) larger round spermatid nuclear diameter than all the other

birds used in the study, except bird numbers 05, 07 and 08. Also, preleptotene primary

spermatocytes in bird number 06 had larger (p < 0.05) nuclei than those found in bird numbers 01,

03 and 10. Bird number 10 on the other hand, had significantly (p < 0.05) bigger type I

spermatocytes nuclei than bird numbers 01, 08 and 09. Similarly, bird number 07 had a bigger type

I spermatocyte nuclear diameter than bird numbers 01, 02, 05, 06 and 08. Bird number 04, however,

had a similar (p > 0.05) type I spermatocyte nuclear diameter to all but bird numbers 01 and 08.

Steps of Spermiogenesis and Cellular Associations in the Seminiferous Epithelium

The steps of spermiogenesis and cellular associations in the seminiferous epithelium of a breeding

guinea cock are shown in figures 1 and 2. Eleven steps of spermiogenesis were identified based on

acrosomic differentiation and nuclear morphological changes.

Steps of Spermiogenesis

Step 1: New generation of spermatids had spherical nucleus, with a narrow band of chromatin

under the nuclear membrane, and containing a number of centrally located heavily stained

chromatin bodies. A faintly staining area representing the Golgi region was evident in the

cytoplasm of the spermatid.

Step 2: These spermatids had round nuclei with chromatin condensed, and adhered to the nuclear

membrane in an uninterrupted manner. A large, centrally located mass of chromatin was seen

connecting to the peripheral clumps by thin filamentous chromatin material. Small intensely stained

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proacrosomic granules were located in the cytoplasm between the nuclear and plasma membranes.

Step 3: Step 3 spermatids had round nuclei, and still had chromatin clumps adhered to the nuclear

membrane and connecting to the centrally located chromatin mass by fewer chromatin strands.

These spermatids are largely distinguished by the attachment of the acrosomic granules to the

nuclear membrane.

Step 4: These spermatids were distinguished by the disappearance of the central chromatin mass,

and the heavy condensation of the nuclear chromatin along the nuclear membrane in an

uninterrupted fashion. The proacrosomic granules increased further and remained attached to the

nuclear membrane.

Step 5: Further condensation of the chromatin material had occurred and proacrosomic material

had become larger than in step 4, thereby increasing its angle of attachment to the nuclear

membrane. The nuclei were round and smaller than all preceding spermatids, and retained the

staining characteristics of preceding steps.

Step 6: Nuclear morphological changes were obvious for the first time. Nuclei generally

appeared pear-shaped with condensed chromatin still attached to the nuclear membrane. Nuclei

seemed to be divided into lobes with the chromatin material adhering firmly along each lobe. Small

area of the nucleus at this point, the spermatid head, was covered by the acrosome, and this was

directed towards the basal lamina.

Step 7: Spermatids had further elongated with considerable cytoplasm. The heavily condensed

chromatin material moved along the nuclear length in a spiral fashion. The nucleus was at this point

divided into further lobes. The tail filament was visible at this stage, and the acrosomic granule

became conical shaped.

Step 8: Nuclei at this stage have further elongated, with condensed chromatin becoming wavier

in shape. The head of the spermatid became tapering at the apex, while the tail end retained a clear

circular vacuole, with considerable reduction in both nucleoplasm and cytoplasm.

Step 9: Maximum length of the spermatid was attained at this stage. There was a further

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elongation of the nucleus accompanied by decrease in wavy appearance of the condensed chromatin

material. The nucleus was within the spermatid cytoplasm, which had further reduced. The

nucleoplasm had virtually vanished at this point. The spermatid head remained pointed, while the

chromatin tail lost its vacuole and had condensed to give rise to the tail filaments.

Step 10: At this point, the chromatin became less spiral. The entire nucleus was absent except the

chromatin material and the acrosome. The cytoplasm had further reduced.

Step 11: This was the final stage prior to sperm release. At this stage, the spermatid was gently

curved and cytoplasm had completely disappeared.

Cellular Associations in the Seminiferous Epithelium of Guinea Fowl

Nine cellular associations were found in the seminiferous epithelium of guinea fowls. Each

association occupied a small area. In some instances, all nine associations were found in a single

tubular cross-section. No tubular cross-section contained only one cellular association. Atypical

associations occurred, usually by spermatids from adjacent associations intruding. Such

associations were not considered in the description of cellular association. Changes in the

morphology of earlier generations of spermatids (steps of spermiogenesis) were used in the

description of cellular associations. Continuous presence of spermatogonial A, B and intermediate

types, as well as leptotene primary spermatocytes were encountered in the seminiferous epithelium.

Stage I: This stage was identified by the presence of earliest generation of spermatids at step 1 of

spermiogenesis. Step 10 spermatids were clustered in groups embedded in Sertoli cell cytoplasm,

with the tails lining the luminal border, and had large mass of cytoplasm. Younger series of

leptotene and zygotene primary spermatocyte were found just at the second and third rows,

respectively, overlaying the spermatogonia.

Stage II: This was distinguished by the presence of spermatids at step 2 of differentiation. All the

other germ cells were as in stage I above.

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Stage III: Step 3 spermatids, as described in steps of spermiogenesis above, were present at this

stage. Mature spermatids were at the stage of spermiation, having lost all their cytoplasm, and

shortened. Leptotene primary spermatocytes were located on the second row, while zygotene cells

occupied the third and fourth rows. Lining the basal lamina were spermatogonia.

Stage IV: This stage was characterized by the presence of step 4 spermatids. The heads of mature

spermatozoa at step 11 lined the luminal border. Spermatogonia lined the basement membrane,

leptotene primary spermatocytes occupied the second row, while pachytene cells occupied between

the third and fourth germ cell layers.

Stage V: The difference between this and the previous stage was the appearance of step 5

spermatids, which had bigger acrosome with wider angle of attachment than step 4 spermatids. All

germ cells remained as in step 4.

Stage VI: This stage was characterized by the emergence of step 6 spermatids, when

morphological changes in the nuclear of round spermatids occurred for the first time, with the

spermatid head oriented towards the basement membrane. Few spermatozoa were present in the

tubular lumen. Other germ cells were arranged in the order, spermatogonia on the basement

membrane, leptotene primary spermatocytes and pachytene primary spermatocytes at subsequent

layers.

Stage VII: At this stage, step 7 spermatids emerged. These spermatids formed about 3 to 6 lobes,

with the chromatin material showing helical migration along the nucleus. Leptotene and pachytene

spermatocytes were present, with spermatogonia on the basement membrane.

Stage VIII: Step 8 spermatids were present, and these were far longer than step 7 spermatids.

The spermatids began to form clusters embedded in Sertoli cells. They looked indistinct because

they were still within the cytoplasm. Both leptotene and pachytene primary spermatocytes were

present. Also, spermatogonia were present on the basement membrane.

Stage IX: At this stage, were spermatids that were less spiral than preceding spermatids. These

were in clusters, and were found at the luminal border of the epithelium. Most of their cytoplasm

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was lost at this stage. Spermatogonia were present along the basal lamina. Leptotene primary

spermatocyte formed the next layer, while diplotene primary spermatocyte at meiotic division and

secondary spermatocytes spanned from the third layer to the spermatids.

To follow the stages of spermatogenesis in the guinea fowl using the cycle map, proceed

horizontally from left to extreme right of a particular row (from the bottom), and then continue from

left to right on the next row up and so forth until the stage during which sperm release is indicated,

as in stage 4 in the last row. This is clearly shown in figure 1.

DISCUSSION

Eleven steps of spermiogenesis and 9 stages of seminiferous epithelium were observed in the

guinea fowl testis. The classification of seminiferous epithelial cycle in the guinea fowl was similar

to those described in other birds and mammals. Also, three spermatogonial types, namely,

spermatogonial A, B and intermediate types were identified.

In epoxy resin embedded toluidine blue stained sections of the guinea fowl testis, the presence of

acrocomic granules facilitated the identification of earlier spermatids (steps 1-5), while nuclear

morphological changes facilitated the recognition of steps 6 to 11 spermatids. Similar observation

was made by Lin et al. (1990) in Japanese quail. Beyond step 5 no noticeable changes in the angle

of attachment of the acrosomic granule to the nuclear membrane were found. This is in agreement

with the reports of Aire et al. (1980) in an earlier study. The author, however, indicated that PAS-

staining became ineffective beyond step 4, and argued that steps of spermiogenesis in the guinea

fowl may even be identified without much difficulty using haematoxylin-eosin stained sections.

Clermont (1958) noted that PAS staining was useful in recognition of up to step 7 spermatids in the

drake, while de Reviers (1971) and Gunawardana (1977) made a similar observation from step 4 in

the domestic fowl. Acrosomal vesicles were not seen in early stages of spermatids. Similar

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observations were made by Aire et al. (1980) in an earlier study on the same species, and

Gunawardana (1977) in the domestic fowl. Similar to the reports of Aire et al. (1980) in the guinea

fowl, Clermont (1958) in drakes and Yamamoto et al. (1967) in the Japanese quail, the cap phase

that characterises mammalian spermiogenesis was absent in all stages of the cycle in the guinea

fowl. In the domestic fowl, however, conflicting observations were made. While an earlier study

(Cavazos and Mellampy 1954) reported cap formation in this species, de Reviers (1971) and

Gunawardana (1977) demonstrated the opposite.

The nucleus of guinea fowl spermatid undergoes morphological transformations from step 6

when it begins to elongate and assume spiral shape, attaining the maximum length at step 9. Steps

of spermiogenesis in the guinea fowl observed in the present study and earlier described by Aire et

al. (1980) are similar to those described by Clermont (1958) in the duck.

The results of the present study revealed a regular, well defined cycle of seminiferous

epithelium in the guinea fowl using epoxy resin embedded sections. The cycle may be divided into

9 stages according to the developmental state of the acrosome (Leblond and Clermont 1952) and the

nuclear morphology of spermatids (Ortavant 1954; Aire et al. 1980). The identification of the

various stages of the cycle was also facilitated by flushing apart of the various stages with

phosphate buffer before fixation (Lin et al. 1990). In contrast to the observations of the present

study, Aire et al. (1980) identified 8 stages of seminiferous epithelium in this species. Ten stages

were reported in the Japanese quail (Lin et al. 1990), while the drake (Clermont, 1958) had 8 stages.

The difference between the classification made by Aire and associates (1980) and the results of the

present study is in step 5 spermatids which were missed by the authors. These had a bigger

acrosome and wider angle of attachment to the nuclear membrane than step 4 spermatids.

The classification of the seminiferous epithelium in the guinea fowl described in the present

study is in agreement with those described in the drake (Clermont 1958) and Japanese quail (Lin et

al. 1990), and is similar to the classification for mammals, especially, primates (Clermont and Antar

1973; Chowdhury and Steinberger 1976). In contrast, the classification by Yamamoto et al. (1967)

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in the Japanese quail disagrees with the classification in the present and other studies (Clermont

1958, drake; Lin et al. 1990, Japanese quail). In their study, the arrangement of germ cells in the

seminiferous epithelium were quite different from those described for mammals (Clermont and

Antar 1973; Chowdhury and Steinberger 1976). For instance, stages IV to VIII contained up to 11

types of germ cells, including 3 generations of spermatids, and secondary spermatocytes were

present in 6 stages of the cycle. Consequently, they concluded that spermatogenesis in the Japanese

quail is fundamentally different from that in mammalian species, including man.

Several cellular associations or stages of seminiferous epithelium were present in any one

cross-section of the seminiferous tubule in the guinea fowl. In some instances, all the stages were

found in a single tubular cross-section. This is similar to the observations made in the duck

(Clermont 1958), Japanese quail (Yamamoto et al. 1967; Lin et al., 1990) and man (Clermont

1963). This is, however, dissimilar to the observations made in most mammals (Johnson et al.

2000). Clermont found atypical or heterogonous cellular associations in man, a phenomenon where

spermatids from adjacent stages may intermix, and thus, produce abnormal associations that cannot

be placed in the normal sequence of the stages of seminiferous epithelium. This phenomenon was

also noticed in the present study, and in an earlier study on the guinea fowl (Aire et al. 1980), duck

(Clermont 1958) and Japanese quail (Yamamoto et al. 1967).

The significant differences in nuclei diameters of spermatocyte I and round spermatids among

individual birds were not surprising, considering the fact that spermatocytes following preleptotene

stage undergo a gradual increase in size up to the first meiotic division (Kerr 2007), while round

spermatids undergo continuous nuclear condensation during maturation, until a fully formed

spermatozoa is extruded into the tubular lumen (Hess and de Franca 2008). In birds with averagely

larger spermatocytes nuclei, therefore, most of the cells sampled might be those at a more advanced

stage of maturation than those with smaller nuclei. Conversely, in birds with averagely larger round

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spermatid nuclei, most of the spermatids sampled would have been at an earlier stage of maturation,

including late telophase cells present at the conclusion of meiosis.

Three types of spermatogonia were identified in the seminiferous epithelium of guinea fowls in

the present study, as opposed to only two reported earlier (Aire et al. 1980). All the three types of

spermatogonia were present in all stages of the cycle and were therefore not distinguished in the

classification. These were named spermatogonial A, B and intermediate types, following the

nomenclature previously used by Yamamoto et al. (1967) in the Japanese quail. Lin et al. (1990)

also found 3 types of spermatogonia in the Japanese quail and named them dark type A, pale type A

and type B spermatogonia, following the nomenclature used by other workers (Clermont 1963;

Clermont and Antar 1973). The types A and B spermatogonia described in the present study

corresponded, respectively, to those described in an earlier study in the guinea fowl (Aire et al.

1980). However, the author did not find the intermediate type in this species. Comparing the results

of this study to those in the quail, the types A, intermediate and B spermatogonia corresponded to

pale type A, dark type A and type B spermatogonia, respectively, described by Lin et al. (1990).

Types A and B spermatogonia in the present study also corresponded to A and B respectively, of

Yamamoto et al. (1967). As noted by Lin et al. (1990), it is uncertain how the intermediate

spermatogonia of Yamamoto et al. (1967) relates to dark type A spermatogonia (intermediate

spermatogonia in the present study). It, therefore, appears that similar spermatogonial types are

found in guinea fowl and Japanese quail.

It is not clear how the three spermatogonial types are related. Nonetheless, the interpretation

of Lin and associates (1990) provides some insight. The authors noted that the intermediate types

are most probably the stem cells in the quail, because they are always located on the basement

membrane, and they are not as mitotically active as the type A cells. Further studies are required to

elucidate the relationship between the 3 spermatogonial types found in guinea fowls and their

renewal.

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DECLARATION OF INTEREST

The authors declare that there is no conflict of interest that could be perceived as prejudicing the

impartiality of the article.

ACKNOWLEDGEMENTS

The authors wish to thank Iain Macmillan and Peter O’Shaughnessy, all of Veterinary Biosciences,

University of Glasgow, for help on histological techniques. Funding for the project was partly

provided by the commonwealth Scholarship Commission in the UK, and Association of

Commonwealth Universities (CSC Ref No: 2009-378).

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Within germ cell differences in nuclear diameters among breeding guinea cocks 1

Bird ID

GERM CELL TYPE {MEAN±SEM (µm)}

Number of

nuclei

Spermatogonia

A

Spermatogonia

B*

Intermediate

spermatogonia

Preleptotene

spermatocyte

Primary spermatocyte

in prophase I

Round spermatids

01 20 6.67 ± 0.82 3.63±0.01ab 4.63±0.07 6.53±0.08

b 5.07 ± 0.05d 3.02 ± 0.03

b

02 20 6.59 ± 0.82 3.60±0.02b 4.67±0.06

6.76±0.13ab

5.14 ± 0.04cd

3.08 ± 0.02b

03 20 6.73 ± 0.82 3.62±0.02ab

4.79±0.05 6.59±0.07b 5.20 ± 0.05

abcd 3.03 ± 0.01

b

04 20 6.56 ± 0.77 3.64±0.03ab 4.69±0.06 6.80±0.09

ab 5.35± 0.06abc 3.02 ± 0.02

b

05 20 6.73 ± 0.72 3.68±0.03ab

4.60±0.05 6.93±0.13ab

5.14 ± 0.05cd

3.08 ± 0.02b

06 20 6.74 ± 0.72 3.74±0.04a 4.74±0.04 7.20±0.11

a 5.19 ± 0.05

bcd 3.17 ± 0.02

a

07 20 6.59 ± 0.36 3.72±0.03a 4.67±0.04 6.70±0.08

ab 5.40 ± 0.04a 3.08 ± 0.01

b

08 20 6.80 ± 0.79 3.71±0.04ab 4.77±0.03 6.74±0.08

ab 5.12± 0.04d 3.07 ± 0.02

b

09 20 6.59 ± 0.81 3.70±0.04 ab

4.66±0.05 6.74±0.07ab

5.17± 0.05cd

3.03 ± 0.01b

10 20 6.68 ± 0.79 3.62±0.03†ab

4.69±0.04 6.63±0.03b 5.38 ± 0.04

ab 3.07 ± 0.03

b

2

*Minor axis of ovoid nuclei were measured.

Means within a column having no superscript in common are significantly (p<0.05) different. SEM: standard error of mean.

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Stages of seminiferous epithelial cycle in the guinea fowl1. Roman numerals indicate the stages of the cycle while arabic

numerals indicate steps of spermatid development. Spermatogonia were not specified as A, B, or intermediate in the various

stages because all three are invariably present at every stage of the cycle. L: leptotene spermatocyte, Z: zygotene spermatocytes,

P; pachytene spermatocyte, D: diplotene spermatocyte in meiotic division and II: secondary spermatocyte

1 Not drawn to scale

Spermatids

Spermatogonia

Spermatocytes

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Fig. 2: Photomicrographs of some representative stages (Stages I-III, VI and IX) of seminiferous epithelial cycle in the guinea fowl.

Numbers show some steps of spermiogenesis. A, B and I represent types A, B and intermediate spermatogonia, respectively. L-

leptotene primary spermatocyte, Z-zygotene primary spermatocyte, P-Pachytene primary spermatocyte, S-Sertoli cell, II-secondary

spermatocytes. Toluidine blue. x 1200.

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