Received: 23/09/2024 Accepted: 26/12/2024 Published: 21/02/2025 1 of 9
https://doi.org/10.52973/rcfcv-e35521 RevistaCientíca,FCV-LUZ/Vol.XXXV
ABSTRACT
The aim of the study was to analyze the ontogeny of the epididymal
and seminiferous epithelium in the testis of Ouled Djellal lambs
throughout postnatal development until puberty. A total of 24 Ouled
Djellal lambs, aged between 1 and 8 months (mos), were used in
this study, with three lambs selected at each mo of age. The lambs
were surgically castrated monthly, and routine histology along with
histomorphometry was performed on the processed epididymal
and testis tissues using AxioVision Rel 4.6 software. Statistical
analysis revealed that testis weight augmented at an accelerated
rate between 4 and 8 mos postnatal, coinciding with signicant
testicular histology modications. These changes included notable
increases in the seminiferous tubules’ diameter and a rise in
testosterone levels. The height of epithelium, as well as luminal and
ductal diameters were found to be statistically different (P<0.05)
among the epididymis’s caput, corpus, and cauda segments, with
the maximum levels recorded during puberty. At 1 and 2 mos of
age, there were two cell kinds present inside the epithelium of
seminiferous: support cells and gonocytes. By 3 mos of age, the
rst spermatogonia were formed from gonocytes, coinciding with
the testicles reaching an average weight of 4 g. Spermatogenesis
was initiated as gonocytes underwent mitosis, giving rise to
progenitors that further differentiated into spermatogonia. Around
4 and 5 mos of age, the seminiferous tubules began to exhibit a
lumen and primary spermatocytes (spermatocytes I). Around the
age of 6 mos, secondary spermatocytes and round spermatids
formed a single row within the seminiferous tubules. At ages 7
and 8 mos, all generations of germ stem cells were present in the
seminiferous tubules. At 8 mos of age, spermatozoa were became
apparent in several segments of the epididymis in the Ouled Djellal
breed, signaling the start of puberty.
Key words: Histomorphometry; Ouled Djellal lamb; puberty;
spermatogenesis; testis; epididymis
RESUMEN
El objetivo del estudio fue analizar la ontogenia del epitelio
epididimario y seminífero en el testículo de los corderos Ouled Djellal
a lo largo del desarrollo postnatal hasta la pubertad. Se utilizaron un
total de 24 corderos Ouled Djellal, con edades comprendidas entre
1 y 8 meses, seleccionando tres corderos en cada mes de edad.
Los corderos fueron castrados quirúrgicamente mensualmente,
y se realizó histología de rutina junto con histomorfometría en
los tejidos epididimarios y testiculares procesados utilizando el
software AxioVision Rel 4.6. El análisis estadístico reveló que el peso
testicular aumentó a un ritmo acelerado entre los 4 y los 8 meses
postnatales, coincidiendo con modicaciones signicativas en la
histología testicular. Estas modicaciones abarcaron incrementos
significativos en la medida de los túbulos seminíferos y un
incremento en los niveles de testosterona. Se encontró que el
diámetro ductal, el diámetro luminal y la altura del epitelio eran
estadísticamente diferentes (P<0.05) entre las regiones caput,
corpus y cauda del epidídimo, con los niveles máximos registrados
durante la pubertad. A los 1 y 2 meses de edad, había dos tipos
de células presentes en el epitelio seminífero: células de soporte
y gonocitos. A los 3 meses de edad, se formaron los primeros
espermatogonios a partir de gonocitos, coincidiendo con el peso
promedio de los testículos alcanzando 4 g. La espermatogénesis
se inició cuando los gonocitos sufrieron mitosis, dando lugar a
progenitores que se diferenciaron en espermatogonios. A los 4 y 5
meses de edad, se observó inicialmente un lumen y espermatocitos
primarios (espermatocitos I) en los túbulos seminíferos. A los
6 meses de edad, espermatocitos secundarios y espermatides
redondas formaron una única la dentro de los túbulos seminíferos.
A los 7 y 8 meses de edad, todas las generaciones de células
madre germinales se encontraban en los seminiferous tubules.
A los 8 meses de edad, se observaron espermatozoides en varios
segmentos del epidídimo en la especie Ouled Djellal, dando inicio
a la etapa de pubertad.
Palabras clave: Histomorfometría; corderos Ouled Djellal;
pubertad, espermatogénesis; testículo; epidídimo
Histological analysis of epididymis and testis in the dynamics of
postnatal ontogenesis from one month to puberty of Ouled Djellal lambs
Análisis histológico del epidídimo y los testículos en la dinámica de la ontogénesis
postnatal desde un mes hasta la pubertad de los corderos Ouled Djellal
Yamina Belkhiri
1,2
* , Farida Bouzebda–Afri
3
, Zoubir Bouzebda
3
, Souheyla Benbia
1,2
, Ramzi Lamraoui
1,3
1
University of Batna 2, Faculty of Natural and Life Sciences, Biology of Organisms Department. Batna, Algeria.
2
University of Batna 2, Biotechnology’s Laboratory of the Bioactive Molecules and the Cellular Physiopathology. Batna, Algeria.
3
Souk–Ahras University, Biotechnologies and Health. Institute Agronomic and Veterinary Sciences, Laboratory of Animal Productions. Souk Ahras, Algeria.
*Corresponding author: y.belkhiri@univ–batna2.dz
Histological analysis of epididymis and testis of Ouled Djellal lambs / Belkhiri et al.________________________________________________
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INTRODUCTION
For domestic animals to have successful reproductive
management, it is crucial to comprehend when puberty and sexual
development begin. The timing of sexual maturity can signicantly
influence an individual’s reproductive efciency. Characterizing
puberty and the initial stages of sexual maturation is crucial for
selecting males within a specic breed, Early sexual maturity
shortens the generation interval and permits early progeny testing,
thereby enhancing the selection process. In this regard, it is
noteworthy that most sheep breeds are seasonal producers, and
that the age at which males reach sexual puberty varies according
to a number of factors, including season, breed, age, body weight,
development of the testes, and the enhanced efcacy of endocrine
gland secretions [1].
In lambs (Ovis aries), gonocytes move centrifugally from their
central position toward the base membrane during the early
postnatal stage, where they transform into spermatogonial stem
cells and produce differentiated spermatogonia [2]. Postnatal
testicular development and function are governed by a complex
interplay of growth factors, cytokines, and gonadotropins,
alongside the establishment of the hypothalamo–hypophyseal–
gonadal axis. The onset of the pre–pubertal period is marked by
the initiation of spermatogenesis, leading to sperm formation due
to the progressive increase in LH pulses. Spermatogenesis is a
systematic process that is ongoing in males, but it doesn’t happen
in every seminiferous tubule at the same time [3].
Instead, it develops in wave–like maturation sequences called
seminiferous epithelium cycles. The rst signs of puberty are the
completion of spermatogenesis and the sperm cells found in the
epididymis or seminiferous tubules [4].
In sheep (Ovis aries), measuring the most fundamental
morphometric traits of the reproductive system is a useful tool for
assessing the breeding soundness and potential fertility of breeding
males. Testicular biometrics, in particular, are crucial for interpreting
spermatogenesis, selecting superior rams, and assessing sexual
maturity throughout postnatal development. Also, Ibrahim et al.
[5] noted that qualitative changes in testicular components and
spermatogenic activities must be assessed and estimated using
testicular morphological examination in any breed or species.
In addition to histological analysis, the study of the epididymis
provides reliable information for determining the stage of maturity.
Anatomically, the epididymis of ovine species is separated into cauda
(tail), corpus (body) and caput (head). It contains the nal segments
of the efferent ducts and features a densely coiled epididymal duct
along its entire length [4]. The epididymis plays crucial roles in sperm
storage, maturation, and absorption [6].
In male sheep, sex hormones are vital for completing the sexual
development process [1]. Testosterone is crucial in initiating sexual
desire in ram lambs, as demonstrated by the rst emergence of
spermatozoa in the ejaculate [7]. Fluctuations in testosterone levels
throughout the postnatal period can have a substantial impact on
the spermatogenesis process as well as the size and weight of the
testicles and accessory reproductive organs [8].
The timing of puberty and sexual maturity, as well as sexual
development have been studied in Blackbelly sheep [2], Corriedale
singleton lambs [4], Ghezel breed rams [9] and Najdi and Naemi
ram lambs [10]. However, similar studies have not yet been
conducted on Ouled Djellal rams, also known as the White Arab
breed or Arbia, which are native to the arid and semi–arid regions
of Algeria. Given the likely signicant variation both between and
among different breeds, a comprehensive study on this aspect of
reproductive function in Ouled Djellal rams is required. Therefore,
it was essential to describe the morphometric characteristics of
the epididymis, as well as the different seminiferous epithelial
cells, both in terms of quality and quantity during prepubertal
development in the Ouled Djellal ram breed. This study focused
on the timing of the development of certain kinds of germ cells,
the commencement of spermatogenesis, and the onset of puberty.
MATERIALS AND METHODS
Animals and location
The investigation was conducted at the Khebbaba farm in
Mezloug (Setif), Algeria. Mezloug is located around 10 km South–
East of Setif in latitude 36°6'28" North and longitude 5°20'13"
East, with a mean altitude of 933 m. The climate in Setif is
semi–arid, characterized by four distinct seasons. The average
temperature ranges from 4.6°C in January to 35°C in July, with
an annual average rainfall of 456 mm.
A total of 24 Ouled Djellal ram lambs were used, with three lambs
at each month of age between 1 and 8 months (mos). Animals
were born in autumn. Throughout the trial the rams were fed a
typical growth ration and had unfettered access to both mineral
block and water. Each lamb’s body weight (BW) was measured at
the beginning of the experiment utilizing a livestock scale tailored
for small ruminants (Marechalle Weighing, France).
Tissue collection and histological procedure for light microscopy
The testes were removed following anesthetic induction and
lateral scrotal incision. Then, for histomorphometric analysis,
the left testes and epididymides were subjected to xation in
a 10% formalin solution, while the right testes were used for
biometric analysis.
The formalin–xed tissues were processed as follows: removal of
the xative with an ascending ethanol series (sequential immersion
in 70%, 95%, and 100% ethanol for 2 h, clearing with xylene
2 h, embedding in parafn, sectioning with a microtome (Leica
RM2125 RT, Germany), mounting 5 µm thick sections on glass
slides, and staining with hematoxylin–eosin. A light microscope
(ZEISS, Axioplan, Germany) with a digital camera (MICROCAM
MA88-500, Germany) was used to take digital images of the testes
and the three parts of epididyma (cauda, corpus, and caput) for
morphometric analysis. The images were examined using the
AxioVision Rel 4.6 software (Carl Zeiss, Thornwood, USA).
The tissue area lled by tubular and interstitial tissue in each
selected eld was quantied using a 40× objective. Seminiferous
tubule diameter was measured at 40× magnification. Thirty
spherical (or almost spherical) tubule proles were selected
and evaluated for each testis in each lamb. The seminiferous
epithelium’s height was also measured using these sections.
Histological analysis of epididymis and testis of Ouled Djellal lambs / Belkhiri et al.________________________________________________
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The weight of both testes (in g) was directly converted into their
volume (VT), as the testicular volume density in mammals is known
to be approximately one, according to Belkhiri et al. [11]. The
relative volume (Vr) of the seminiferous tubules, calculated as the
area occupied by the tubules divided by the total area of the eld.
The total seminiferous tubule volume per testis (VTS) was then
obtained by multiplying Vr by VT. To determine the seminiferous
tubules’ total length (LST) (m), the tubules were modeled as a
single cylinder with radius r and a volume of seminiferous tubules
(VTS). Equation LST=VTS/π r
2
is obtained by using the formula
VTS=π r
2
LST; Where: π = 3.14.
In the epididymis, each region’s ductal diameter (caput, corpus,
and cauda) was measured by randomly selecting and analyzing
20 ductal cross–sections per animal, ensuring the sections
were as round as feasible. Additionally, the epithelial height and
luminal diameter were measured in these sections. The horizontal
distance, excluding the stereocilia, between the epithelium’s base
(basal lamina) and its apical border was used to describe the
epithelium’s height. The greatest distance between two apical
margins across the lumen was measured as the luminal diameter,
and the maximum distance across basal laminas was measured
as the tubular diameter [12].
Each lamb’s testis at each age was used to investigate 30 cross–
sections of seminiferous cords and tubules in order to study the
development of postnatal germ cell populations.
As described by Bahaodini et al. [13] various spermatogenic cells,
such as support cells, Sertoli cells, gonocytes, spermatogonia,
primary spermatocytes (leptotene, zygotene, pachytene, and
diacinese stages), secondary spermatocytes, and spermatids
(round and elongated), were categorized based on particular
morphological. This evaluation was performed at 100×.
Blood concentration of testosterone
From one month old until puberty, blood samples were taken
every month from the external jugular vein to measure plasma
testosterone levels. Following a 10 min centrifugation (Thermo
Scientic Heraeus® Labofuge® 200, Germany) at 1500 × g, the
samples were decanted and stored at -20°C (Thermo Scientic™
Forma, Germany) until analysis. Testosterone levels were measured
using electrochemiluminescence immunoassay (ECLIA).
Statistical analysis
Values are expressed as the mean with the standard error of the
mean (SEM). Differences in weight measurements, testosterone
levels, and histomorphometric values across diverse postnatal ages
were assessed using one–way variance analysis (ANOVA), followed
by a post–hoc analysis using Tukey’s HSD test, with statistical
analysis performed using SPSS (Ver. 2023). Signicant differences
were considered as P–values of 0.05 or below.
RESULTS AND DISCUSSION
This study is the rst to describe the development of testicular
parenchyma and epididymal epithelium in Ouled Djellal ram
lambs during postnatal growth. In each animal, sexual maturity
was marked by the presence of spermatozoa in the epididymal
lumen [5, 6]. Understanding and mastering male reproduction is
a cornerstone in breeding and management programs.
Age–related changes in weight measurements
Table I shows variations in total weight, testicular weight, and
epididymis weight with age. The ndings of the current study
showed notable monthly variations in body weight (BW) (P<0.05)
of Ouled Djellal ram lambs from the rst (9.60 ± 0.36 kg) to the
eighth month (43.80 ± 0.48 kg) (TABLE I). These ndings are
largely consistent with data from other studies [9, 10], with minor
differences likely attributable to breed and environmental factors
in which the rams were raised. Body weight variations can likely
be attributed to differences in overall body development, which
reflect anabolic processes and growth rates. Additionally, hormonal
influences, particularly from growth hormone and other hormones
linked to body mass development, as well as differences in
genotype, may also contribute signicantly to these variations [14].
These results showed that testicular and epididymal
development in Ouled Djellal rams was slower (P<0.05) in the
initial postnatal period and accelerated from 4 to 8 mos of age
(P<0.001), approximately the time when spermatogenesis begins.
Therefore, the 4 mos of age marks a crucial period in the pubertal
development of Ouled Djellal ram lambs. In selecting males of this
breed, testis weight is signicantly more relevant than body weight.
This nding supports previous observations documented in
various ram breeds, including Arrabi and Awassi sheep males
[14]. The rst sluggish period of testicular growth is marked by
low blood testosterone levels, but the ensuing fast growth phase
is marked by much greater testosterone concentrations. This
hormonal shift plays a critical role in accelerating testicular growth
and the onset of spermatogenesis [15]. Wańkowska [16] identied
another factor in her research on sheep, noting that increased body
weight correlates with heightened secretion of metabolic hormones
like growth hormone, which directly affects the growth of body
organs, including the testes. Other studies have demonstrated that
TABLE I
Weight measurements and testosterone levels in
Ouled Djellal lambs at dierent ages
Age
(months)
Parameters
Epididymis
weight (g)
Testicular
weight (g)
Body weight
(kg)
Testosterone
(ng·ml
-1
)
1 0.51 ± 0.02
a
1.30 ± 0.11
a
9.60 ± 0.36
a
0.07 ± 0.01
a
2 1.02 ± 0.05
ab
2.40 ± 0.19
ab
16.80 ± 0.73
bc
0.09 ± 0.00
a
3 2.18 ± 0.32
b
4.00 ± 0.15
b
22.80 ± 0.48
c
0.11 ± 0.01
a
4 5.16 ± 0.17
c
15.80 ± 1.95
c
29.80 ± 0.73
d
0.12 ± 0.07
a
5 10.03 ± 0.43
d
20.60 ± 2.20
d
33.00 ± 0.00
de
0.20 ± 0.00
b
6 21.50 ± 0.15
e
58.40 ± 0.97
e
35.60 ± 0.24
ef
0.31 ± 0.03
c
7 29.32 ± 0.47
f
77.00 ± 0.01
f
38.40 ± 0.24
fg
0.50 ± 0.02
d
8 38.98 ± 0.13
g
99.40 ± 0.24
g
43.80 ± 0.48
h
0.58 ± 0.03
e
Thedierencesbetweenageswithincolumnsareindicatedbydierentsuperscripts
a,b,c,d,e,f,
g
(P<0.05)
Histological analysis of epididymis and testis of Ouled Djellal lambs / Belkhiri et al.________________________________________________
4 of 9 5 of 9
a quick increase in testicular weight coincided with elevated serum
testosterone concentrations, as well as enhanced gonadotropin
receptor concentrations and afnity. Li et al. [17] demonstrated
that high levels of postnatal serum gonadotropins and testicular
gonadotropin receptors are believed to have triggered testicular
development. This growth was sustained by the increased
responsiveness of Leydig and Sertoli cells to reduced blood FSH
and LH concentrations, as observed in male sheep.
Age–related changes in testosterone levels
TABLE I shows the variations in testosterone levels with age.
According to these ndings, there were no signicant changes
in testosterone levels between one and four months (P>0.05).
Low testosterone levels throughout the neonatal phase do not
inhibit spermatogonia formation, during the early stages of
spermatogenesis is unaffected by androgens and gonadotropins.
Indeed, Sertoli cells do not have receptors for androgenic hormones
during the majority of the early neonatal period [18]. Zirkin and
Papadopoulos [19] demonstrated in rats that the early infantile
period is characterized by relatively stable yet reduced testosterone
levels. This is a result of the progressive decline of fetal Leydig
cells and the ongoing development of mitotically active progenitor
Leydig cells, which predominantly synthesize dihydrotestosterone
(DHT) and 3α–androstanediol, rather than testosterone [20]. These
results demonstrated a sudden and rapid increase (P<0.05) in
testosterone levels among Ouled Djellal lambs between 4 mos
(0.18 ± 0.01 ng·ml
-1
) and 8 mos of age (0.58 ± 0.01 ng·ml
-1
). These
results are comparable to those of
Al–kawmani et al. [10], who
observed an increase in testosterone concentration between 5
and 9 mos of age in Najdi and Naemi sheep. Zornitzki et al. [21]
showed that the increase in testosterone levels can be attributed
to various factors, including species, breed, age, environmental
conditions, and seasonal variations.
Age–related changes in testicular histology
At 1 and 2 mos of age, the testicular parenchyma was consisted
of several seminiferous cords. Every single sex cord was encircled
by a unique basement membrane and many rows of elongated
peritubular cells called myoid cells. Many stromal or interstitial
cells, including Leydig cells, were seen in the stroma in the areas
between the sex cords (FIG. 1). This result agrees with Elzoghby
et al. [22] in lamb. Only two types of cells were observed in the
seminiferous cords: the majority were supporting cells, which were
arranged peripherally and perpendicular to the base membrane.
The other type of cells was the gonocytes, which had larger nuclei
compared to the supporting cells. Gonocytes were arranged near
or even within the wall of the supporting cells’ nuclei.
Figure 1. Histology of testes from Ouled Djellal lambs at dierent ages. Sections (5 µm thick) of testes stained with Hematoxylin and Eosin. (A) and
(B) At 1 and 2 months of age, the seminiferous tubules were composed of supporting cells (blue arrow) and gonocytes (red arrow). Each sex cord was
surrounded by a distinct basement membrane (black arrow), and The seminiferous tubules were divided by interstitial tissue rich in Leydig cells (green
arrow) (100× magnication). (C) At 3 months of age, the seminiferous tubules consisted of spermatogonia (red arrow) and Sertoli cells (blue arrow)
(100× magnication). (D) and (E) At 4 and 5 months of age, the seminiferous tubules contained spermatogonia (red arrow), Sertoli cells (blue arrow),
leptotene spermatocytes (brown arrow), zygotene spermatocytes (yellow arrow), pachytene spermatocytes (violet arrow), and diplotene spermatocytes
(orange arrow) (40× magnication). (F) At 6 months of age, the seminiferous tubules included all of the previously mentioned cells, with the addition
of secondary spermatocytes (white arrow) and round spermatids (grey arrow) (40× magnication). (G) At 7 months of age, the seminiferous tubules
contained all the congurations marking the dierent stages of spermatogenesis, though in very small numbers(40× magnication). (H) At 8 months
of age, the seminiferous tubules showed completely mature spermatogenesis (100× magnication)
Histological analysis of epididymis and testis of Ouled Djellal lambs / Belkhiri et al.________________________________________________
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5 of 9
At 3 mos of age, spermatogonia are the offspring cells
of gonocytes that enter mitosis, marking the beginning of
spermatogenesis when the testicle reaches an average weight
of about 4 g. This founding was similar to that in the Blackbelly
breed (9-12 week) [2], however, considerably later than in the Nadji
breed lamb (2 mos) [23] and in Ghazel breed lamb (1 month) [9].
The beginning of spermatogenesis may differ across lamb breeds.
Increased serum FSH concentrations, mediating effects via FSH
receptors located on Sertoli cells, enhance the transformation from
from gonocytes into spermatogonia [24]. Sertoli cell differentiation
in Ouled Djellal lambs began around the time of spermatogenesis
and lasted around 2 mos. These early differentiated Sertoli cells
supported the earliest steps of spermatogenesis, which included
the differentiation of early spermatogonia and the subsequent
development of germ cells during meiosis during the rst wave
of the cycle. In bull calves, testosterone has been proposed to
induce the differentiation of indifferent supporting cells (immature
Sertoli cells) [20, 25].
At four months of age, primary spermatocytes begin to emerge,
corresponding to a testicular weight of 15.8 g, but they were few
and present in only a few tubules. One month later (at 5 mos), with
a testicular weight of 20.6 g, primary spermatocytes were present
in all tubules. In contrast, pachytene spermatocytes were found in
the majority of tubules at 12 weeks earlier than the age at which the
quantity of sertoli cells were constant (15 weeks) [2]. Santi etal.
[26] demonstrated that mean serum FSH concentrations and FSH
receptor (FSH–R) levels were elevated when the immature germ
cells, occupied the majority of the seminiferous tubules. This most
likely helped these immature germ cells proliferate and differentiate
into primary spermatocytes in response to FSH. The lumen of the
seminiferous tubules appears when the central cells disintegrate
and the intercellular material of the sexual cords liquees. This
occurred at 4 mos in the Ouled Djellal lambs, which was similar to
that of the Ghezel breed rams [9]. At this postnatal age, we detected
a sharp and signicant rise in the tube diameter of Ouled Djellal
rams. The lumen formation, occurring mainly after the placing of
the blood–testis barrier, in parallel to the production of fluid from
the sertoli cell, as well as the transport of fluid in interstitial tissue
by Sertoli cells [27].
In the present study, secondary spermatocytes rst appeared
at 6 mos of age. According to Oduwole et al. [28], increased LH
receptor activity may have stimulated the LH–dependent secretion
of testosterone, which is necessary for the development of primary
spermatocytes into secondary spermatocytes. The age at which
spermatids were rst observed was 6 mos, which was similar to
that of the Black Bengal goats [29], and was earlier than the Nadji
lambs (7 mos) [23] and later than the Blackbelly lambs (5 mos) [2].
Rajak et al. [20] found that insufcient androgen levels result in an
immediate halt in the meiotic transition of primary spermatocytes
to spermatids, thereby inhibiting sperm production.
At 7 mos of age, the histological appearance was indicative
of sexually mature sheep. The seminiferous tubules exhibited
congurations marking different stages of spermatogenesis, but in
very small numbers (FIG. 1). All the lumina of the tubules were empty.
These results indicated that spermatozoa were present in the
epididymal lumen, as revealed by histological studies, suggests
that Ouled Djellal rams reach puberty at around 8 mos of age.
This nding is inconsistent with other studies on the same breed
that estimated the onset of puberty based on the rst detection
of sperm in ejaculate, which occurred at approximately 228 days
[30]. In contrast, Blackbelly sheep had spermatozoa in the various
segments of the epididymis at 18-21 weeks of age, indicated
the beginning of puberty [2]. Environmental factors affect sexual
development in lambs, with the season of birth influencing the
time between puberty and sexual maturity. Lambs born in the
autumn exhibit faster testicular growth compared to those born in
the spring. Nutrition is another key environmental factor impacting
the onset of puberty [31].
Age–related changes in the numbers of germ cells, Sertoli cells,
and Leydig cells
The results of different germ cells number at different age of
lambs are presented in TABLE II. The mean count of indifferent
supporting cells (immature Sertoli cells) per testis increased
between 1 and 3 mos of age (P<0.05). As these cells differentiated
into mature Sertoli cells, the average Sertoli cell count per testis
increased until 4 mos of age, then gradually declined until 8 mos
(P<0.05). Our results revealed that the reduced proliferation
of Sertoli cells after 4 mos of age supports the hypothesis that
these cells cease dividing to promote the development of the
seminiferous epithelium during the initial wave of spermatogenesis.
This cessation of mitotic activity coincides with several key events:
an increase in Sertoli cell nuclear volume, the initiation of tubular
fluid secretion, lumen formation, the emergence and widespread
proliferation of primary spermatocytes, and the development of
the Sertoli cell barrier and cytoskeleton [32]. During the foetal
and neonatal periods, thyroid hormone receptors are extensively
expressed on Sertoli cells, with T3 playing a vital role in controlling
Sertoli cell proliferation and, more crucially, maturation [33]. Since
each Sertoli cell has a limited capacity to support germ cells, the
number of Sertoli cells in the adult testis determines both testicular
size and daily sperm production.
These results showed that the number of gonocytes/
spermatogonia per testis remained low from 1 to 3 mos of age
(P>0.05). A signicant increase in the germ cell population occurred
after 4 mos of age (P<0.05). After 7 mos of age, the number of
spermatogonia showed a propensity to stabilize (P>0.05). This
process has also been reported in other breeds, such as Ghezel,
at 4 mos of age [9]. The number of primary and secondary
spermatocytes, round and elongated spermatids progressively
increased with age once they rst appeared.
The results showed that from the 1
st
to the 4
th
month of age,
the pattern of Leydig cell numbers and serum testosterone levels
was similar (TABLES I and II). After 4 mos, the amount of Leydig
cells in each testis increased, reaching 3.20 × 10
9
by 8 mos of
age. This phase of development seems to be reflected in the
elevated testosterone production observed from 4 to 8 mos of
age. Baguetal., [25] suggested that higher testosterone production
in bull calves is either a result of increased cellular synthesis or
a rise in the number of Leydig cells. Zirkin and Papadopoulos
[19] showed that mature adult Leydig cells have a much higher
capability for testosterone release as they acquire more organelle
components required for steroid synthesis and exhibit greater
responsiveness to circulating LH.
Histological analysis of epididymis and testis of Ouled Djellal lambs / Belkhiri et al.________________________________________________
6 of 9 7 of 9
Age–related changes in testicular morphometric parameters
TABLE III presents data on the total volume of seminiferous
tubules, their total length and diameter, and the volume of the
interstitium during testicular development from 1 to 8 mos of age.
The volume percent lled by the seminiferous tubule varied
considerably across the eight groups (P<0.05). The volume of
seminiferous tubules varied from 38.23 ± 3.45 to 86.70 ± 1.81 %.
A similar nding was observed in Assam goats [34]. The increase
in the area occupied by seminiferous tubules within the testis is
associated with testicular growth, suggesting that the parenchyma
expands more rapidly to produce sufcient spermatozoa for breeding
rams at puberty. We also observed a steady decrease in interstitial
volume, which contrasted with a gradual increase in seminiferous
tubule occupancy within the parenchyma. A similar decrease in
intertubular space was recorded in Black Bengal goat [29].
The diameter of seminiferous cords/tubules is an excellent
parameter for evaluating the progression of spermatogenesis
during postnatal testis development, as well as the maturation
of Sertoli cells and their fluid secretion, which leads to lumen
formation [35]. These results demonstrated that throughout
postnatal development until puberty, both the diameter and length
of the seminiferous cords/tubules steadily increased (TABLE III).
A similar nding was reported in Blackbelly sheep [2] and Black
Bengal goat [29]. In the early weeks after birth, the only germ cells
connected with Sertoli cells were gonocytes and spermatogonia
[32]. Since spermatogonia were located near the basal membrane
and more mature germ cells were absent, the elongation of the
tubules during this period was primarily caused by spermatogonia
and Sertoli cells mitosis. However, as spermatogenesis initiated,
additional germ cell types emerged and proliferated, leading to a
signicant increase in tubule diameter. This enlarged parenchyma
is due to a signicant increase in the diameter of the seminiferous
tubules around puberty. After 4 mos of age, our study found a
substantial rise in tubular diameter in Ouled Djellal rams, indicating
rapid tubule development at the onset of spermatogenesis and
prior to puberty.
Age–related changes in the histmorphometry of the epididymis
This study’s microscopic analysis of the epididymis showed
that the ductus epididymis at 1, 2, and 3 mos of age was lined by
simple epithelial cells, which might be cuboidal or columnar in
shape. These cells’ nuclei ranged from circular to oval and were
characterized by the presence of minute microvilli (stereocilia)
(FIG. 2). At 4 and 5 mos of age, the ductus epididymis was covered
by pseudostratified epithelium that included microvilli. This
epithelial layer was made up of tall principal cells with elongated,
narrow nuclei, as well as smaller basal cells with round to oval
nuclei. At 6 mos of age, pseudostratied columnar epithelium
with more prominent microvilli surrounded the ductus epididymis.
By 7 mos of age, the ductus epididymis was characterized by tall
columnar epithelium with elongated microvilli and an appearance
of near maturity. At 8 mos of age, numerous spermatozo a were
appeared accumulating in the cauda, corpus, and caput epididymis
lumens, with no signicant age–related histological modications
noted in the epididymis. These results align with those found by
TABLE II
Number (Mean ± SE) of dierent cell types per testis in Ouled Djellal lambs at dierent ages
Age
(months)
Total number per testis (× 10
9
)
Supporting
cells /
Sertoli cells
Gonocytes /
Spermatogonia
Primary
Spermatocytes
Primary
Spermatocytes
L
Primary
Spermatocytes
P
Primary
Spermatocytes
Z
Primary
Spermatocytes
D
Secondary
Spermatocytes
Round
Spermatids
Elongated
Spermatids
Leydig cells
1 1.58 ± 0.12
a
0.06 ± 0.01
a
– – – – – – – – 0.49 ± 0.02
a
2 4.01 ± 0.68
b
0.10 ± 0.02
a
– – – – – – – – 0.51 ± 0.01
a
3 7.76 ± 1.22
c
0.13 ± 0.03
a
– – – – – – – – 0.52 ± 0.00
a
4 24.92 ± 6.58
d
0.49 ± 0.18
b
2.62 ± 1.04
a
1.23 ± 0.56
a
0.47 ± 0.17
a
0.90 ± 0.31
a
– – – – 0.58 ± 0.00
a
5 23.37 ± 9.47
de
0.93 ± 0.38
c
6.75 ± 2.65
b
2.59 ± 1.09
b
0.81 ± 0.23
b
2.45 ± 0.99
b
0.87 ± 0.35
a
– – – 0.79 ± 0.01
c
6 19.38 ± 4.68
f
1.62 ± 0.39
d
14.85 ± 3.98
c
3.80 ± 0.85
c
2.34 ± 0.71
c
4.65 ± 0.51
c
2.40 ± 0.66
b
4.73 ± 1.29
a
19.42 ± 4.74
a
– 0.89 ± 0.10
d
7 12.05 ± 1.79
g
1.33 ± 0.23
be
15.86 ± 2.79
cd
4.16 ± 0.53
d
4.90 ± 1.00
d
4.61 ± 0.55
c
2.55 ± 0.42
bc
5.98 ± 1.05
b
22.86 ± 3.83
b
8.33 ± 2.04
a
2.14 ± 0.05
e
8 6.43 ± 0.91
ce
1.32 ± 0.11
be
18.52 ± 1.73
e
4.60 ± 1.13
de
5.72 ± 0.77
e
5.49 ± 1.50
d
3.91 ± 0.44
d
10.34 ± 1.63
c
27.93 ± 3.26
c
63.77 ± 6.69
b
3.20 ± 0.17
f
Thedierencesbetweenageswithincolumnsareindicatedbydierentsuperscripts
a,b,c,d,e,f,g
(P<0.05).
TABLE III
Morphometric values (Mean ± SE) of the testicular
seminiferous tubules in Ouled Djellal lambs
Age
(month)
Parameters
Total seminiferous
tubule volume
per testis (%)
Seminiferous
tubules
diameter (μm)
Length of
seminiferous
tubules (m)/testis
Interstitium
volume (%)
1 38.23 ± 3.45
a
42.04 ± 3.06
a
495.19 ± 83.30
a
61.76 ± 3.45
h
2 43.86 ± 2.53
b
45.55 ± 1.85
a
696.28 ± 90.17
b
56.13 ± 2.53
fg
3 46.47 ± 2.16
bc
54.615 ± 3.71
b
986.88 ± 91.57
c
53.52 ± 2.16
f
4 55.57 ± 2.06
d
78.11 ± 13.86
c
3293.80 ± 269.84
d
44.42 ± 2.06
e
5 74.15 ± 1.55
e
91.78 ± 9.31
d
3304.71 ± 356.42
e
25.84 ± 1.55
cd
6 77.63 ± 1.89
ef
112.94 ± 15.20
de
3431.75 ± 183.76
ef
22.36 ± 1.89
c
7 80.90 ± 1.56
g
150.39 ± 4.69
f
3680.63 ± 333.59
g
19.09 ± 1.56
b
8 86.70 ± 1.81
h
176.96 ± 10.03
g
3730.11 ± 597.83
h
13.29 ± 11.81
a
Thedierencesbetweenageswithincolumnsareindicatedbydierentsuperscripts
a,b,c,d,e,f,g
(P<0.05)
Histological analysis of epididymis and testis of Ouled Djellal lambs / Belkhiri et al.________________________________________________
_________________________________________________________________________________________________Revista Cientica, FCV-LUZ / Vol.XXXV
7 of 9
Bielli et al. [4] in Corriedale singleton lambs. At puberty, mature
spermatozoa predominantly appeared in the cauda epididymis,
with fewer instances observed in the corpus and caput epididymis.
This nding aligns with previous studies indicating that the cauda
epididymis of animals has a higher concentration of sperm [35].
Table IV presents the average measurements of the duct
diameter, lumen diameter, and epithelial height across various
segments of the epididymis in the examined Ouled Djellal ram
lambs. Throughout all three regions, a gradual increase in tubular
diameter, luminal diameter, and epithelial height was observed
with age (P<0.05), becoming more pronounced during the pubertal
stages. These morphometric changes were especially signicant in
the corpus and cauda regions, with the most substantial alterations
occurring in the cauda (P<0.05). Given that the epididymal ducts
play a crucial part in sperm maturation, which favorably affects
sperm morphology, their parietal growth may have improved
their functioning.
CONCLUSION
The postnatal developmental investigation revealed that the
testis in rams grows smoothly and progressively, as shown by
weight measurements and histomorphometric characteristics.
Figure 2. Histological Development of Epididymis in Ouled Djellal Lambs at Dierent Ages. Sections (5 µm thick) of testes stained with Hematoxylin and Eosin. (A)
(B) (C) At 1, 2, and 3 months of age: simple cuboidal to columnar epithelial cells with minute microvilli (stereocilia) were the constituents of the ductus epithelium
(blue arrow head). A muscular layer of smooth muscle (red arrow head) surrounded the duct. The tube itself was encased by connective tissue (white arrow head)
(10× magnication). (D) (E) At 4 and 5 months of age, there were some microvilli and pseudostratied columnar epithelium (green arrow head) lining the ductus
epididymis (10× magnication). (F) The pseudostratied columnar epithelium with prominent microvilli lined the ductus epididymis at six (6) months of age (4×
magnication). (A) At 7 months of age, the ductus epididymis had almost mature look due to its highly columnar epithelial composition (20× magnication).
(H) At 8 months of age, the completely formed epithelium included a ductus epididymis with with abundant mature sperms in the lumen (40× magnication)
TABLE IV
Morphometric parameters of the epididymis in Ouled Djellal lambs at dierent ages
Age
(month)
Parameters
Tubular diameter (µm) Luminal diameter (µm) Epithelial height (µm)
Caput Corpus Cauda Caput Corpus Cauda Caput Corpus Cauda
1 52.32 ± 9.45
a.1
70.58 ± 8.43
a.2
82.51 ± 7.87
a.3
39.25 ± 11.98
a.1
40.25 ± 10.25
a.1
44.91 ± 11.23
a.3
7.11 ± 2.34
a.1
9.42 ± 1.92
a.2
10.11 ± 2.34
a.2
2 66.48 ± 10.98
b.1
89.25 ± 9.34
b.2
98.65 ± 10.98
b.3
40.66 ± 12.02
a.1
46.43 ± 11.36
ab.2
54.18 ± 10.45
b.3
8.28 ± 3.25
a.1
10.65 ± 1.98
a.2
13.08 ± 2.24
b.3
3 70.32 ± 10.76
bc.1
90.98 ± 10.67
b.2
100.25 ± 12.34
b.3
50.36 ± 11.36
b.1
58.32 ± 12.25
c.2
64.75 ± 12.98
c.3
11.12 ± 1.94
b.1
13.93 ± 2.87
b.2
15.90 ± 2.98
c.3
4 77.74 ± 11.76
c.1
96.43 ± 12.43
c.2
118.32 ± 12.98
c.3
59.39 ± 12.08
c.1
67.25 ± 12.98
d.2
74.60 ± 13.76
d.3
15.58 ± 2.45
c.1
16.34 ± 2.34
c.1
17.98 ± 2.67
cd.3
5 87.45 ± 12.67
cd.1
103.43 ± 12.09
d.2
124.98 ± 14.65
cd.3
75.98 ± 14.25
d.1
78.36 ± 14.62
e.2
88.74 ± 13.45
e.3
21.57 ± 2.98
d.1
18.32 ± 1.98
ce.2
22.57 ± 1.98
e.3
6 90.85 ± 13.96
d.1
120.78 ± 13.76
e.2
198.36 ± 14.23
e.3
89.96 ± 15.21
e.1
90.27 ± 14.79
f.1
94.28 ± 14.34
f.3
24.73 ± 1.00
de.1
26.39 ± 2.78
f.2
31.73 ± 3.02
f.3
7 127.14 ± 15.67
e.1
134.98 ± 15.32
f.2
234.54 ± 15.17
f.3
105.98 ± 15.85
f.1
115.74±26
g.2
122.07 ± 15.98
g.3
31.28 ± 3.01
f.1
29.09 ± 2.98
g.2
37.28 ± 2.87
g.3
8 166.70 ± 14.45
f.1
187.34 ± 13.57
g.2
266.70 ± 15.43
g.3
169.25 ± 16.25
g.1
172.36 ± 14.25
h.2
228.03 ± 18.45
gh.3
30.94 ± 2.90
f.1
43.52 ± 3.23
h.2
46.14 ± 3.03
h.3
Thedierencesbetweenageswithincolumnsareindicatedbydierentsuperscripts
a,b,c,d,e,f,g,h
(P<0.05).Dierencesbetween
regionsoftheepididymiswithinarowareindicatedbydierentsuperscriptnumbers
1,2,3
(P<0.05)
Histological analysis of epididymis and testis of Ouled Djellal lambs / Belkhiri et al.________________________________________________
8 of 9 9 of 9
At 4 months of age, there was a large rise in testicular and
epididymal weight, as well as layering of the epithelium of the
seminiferous tubules, a noticeable elevate in seminiferous tubule
diameter, and higher testosterone levels. This represents the
seminiferous tubule lumen’s development. By 8 months of age,
the seminiferous epithelium had completely matured, with all
varieties of spermatogenic cells present, including spermatozoa in
the lumen of the seminiferous tubules and epididymis, indicating
the onset of puberty.
Conflict of interest
No potential conflict of interest was reported by the authors.
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