DOI: https://doi.org/10.52973/rcfcv-e32157
Received: 02/06/2022 Accepted: 30/07/2022 Published: 28/09/2022
1 of 8
Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32157, 1 - 8
ABSTRACT
The objective of this research was to evaluate the effect of sperm on
chromatin stability and its relationship with the membrane integrity
structural – physiological and the rate of fertilization of female sheep.
Ejaculates of sperm (2×10
9
sperm·mL
-1
) with 70% of motility were
collected using an articial vagina (n=5, 2 years old. For this, each
ram was served with fteen female sheep (n=75), generating thus
ve different Groups (A, B, C, D, and E). A control Group also was
considered. Sperm nuclear chromatin stability (NCS) was evaluated
using the Borate Buffer (BB), Sodium Dodecyl Sulfate (SDS), and
the mixture of Ethylenediaminetetraacetic acid (EDTA) + SDS. The
fertilization rate was evaluated after 16-18 hours post sperm injection.
Sperm concentration showed a significant difference (P<0.05)
between Groups. In Contrast, seminal volume, and sperm motility
do not show a signicant difference (P>0.05). A high correlation
(r
2
=0.52) was observed between morphology and motility, and the
fertilization rate was 74.6% (n=56). It was concluded in general that
techniques to evaluate nuclear condensation values do have a high
likelihood to give a diagnosis about the future potential of sperm
populations in Junín ram.
Key words: Sperm chromatin; membrane integrity; Peruvian rams
RESUMEN
El objetivo de esta investigación fue evaluar el efecto de los
espermatozoides sobre la estabilidad de la cromatina y su relación con la
integridad de la membrana estructural – siológica y la tasa de fertilización
de las ovejas hembras. Las eyaculaciones de espermatozoides (2×10
9
espermatozoides·ml
-1
) con un 70% de motilidad se recogieron
mediante una vagina articial (n=5, 2 años). Para ello, cada carnero
se servía con quince ovejas hembra (n=75), generando así cinco grupos
diferentes (A, B, C, D y E). También se consideró un grupo de control.
La estabilidad de la cromatina nuclear (NCS) de los espermatozoides
se evaluó utilizando el tampón de borato (BB), el dodecil sulfato de
sodio (SDS) y la mezcla de ácido etilendiaminotetraacético (EDTA)
+ SDS. La tasa de fertilización se evaluó después de 16-18 horas
después de la inyección de espermatozoides. La concentración de
espermatozoides mostró diferencias signicativas (P<0,05) entre
los grupos. Por el contrario, el volumen seminal y la motilidad de los
espermatozoides no mostraron diferencias signicativas (P>0,05). Se
observó una alta correlación (r
2
=0,52) entre morfología y motilidad, y
la tasa de fecundación fue del 74,6% (n=56). Se concluyó en general
que las técnicas para evaluar los valores de condensación nuclear
tienen una alta probabilidad de dar un diagnóstico sobre el potencial
futuro de las poblaciones de espermatozoides en carneros de Junín.
Palabras clave:
Cromatina espermática; integridad de la membrana;
carneros peruanos
Sperm chromatin stability and their relationship with fertilization rate in
Sheep of the Junín race
Estabilidad de la cromatina espermática y su relación con la tasa de fecundación en ovejas de la raza
Junín
Ide Unchupaico-Payano
1
* , Alberto Alponte-Sierra
1
, Carlos Quispe-Eulogio
2
, Edith Ancco-Goméz
2
, Alex Huamán-De La Cruz
1,4
,
Julio Mariño-Alfaro
1
, Alberto Patiño-Rivera
1
, Carmencita Lavado-Meza
1
, Lupe Huanca-Rojas
1
y Luis Bazán-Alonso
3
1
Universidad Nacional Intercultural de la Selva Central Juan Santos Atahualpa, Vicepresidencia de Investigación. La Merced, Perú.
2
Universidad Peruana Los Andes, Escuela Profesional de Veterinaria y Zootecnia. Chorrillos, Huancayo, Perú.
3
Universidad Nacional del Centro del Perú, Facultad de Ingeniería de Sistemas. El Tambo, Huancayo, Perú.
4
Universidad Peruana Unión, Escuela Profesional de Ingeniería Ambiental. Lurigancho, Lima. Perú.
Email: ide_85@hotmail.com
Sperm chromatin stability in Sheep of the Junín race/ Unchupaico-Payano et al. ______________________________________________________
2 of 8
INTRODUCTION
Infertility is a signicant concern not only in humans but also in
several species of animals [18]. In farm animals, infertility can cause
negative effects on animal welfare and considerable economic loss
[8]. Infertility is related to various causes such as physiological
disturbances, infectious causes, and bad nutrition, which may work
in combination or separately [2, 28]. Likewise, infertility may arise due
to damage to Deoxyribonucleic acid (DNA) and high levels of loosely
packaged chromatin [9]. As sperm cells have the important mission
of delivering vehicles for chromatin cargo within, the oocyte, which is
composed of paternal DNA information and its associated proteins,
its integrity is considered a keystone of reproductive success [31].
Therefore, a correct chromatin package level is necessary for
successful insemination [32]. In the last decades were reported that
chromatin sperm stability contributes to both adequate embryonic
development and successful fertilization [9].
The variability between species is attributed to the proportion of
protamine; in contrast, sperm from the bull (Bos taurus), Rat (Rattus
novergicus), ram (Ovis aries), pig (Sus scrofa domesticus), and guinea pig
(Cavia porcellus), have only one type of protamine (P1), while humans have
a second (P2), which is decient in cysteine residues [1]. In addition,
membrane integrity is very important for metabolism, sperm training,
the acrosomal reaction, and the binding of sperm to the surface of
the oocyte. Plasma membrane damage can cause loss of normal
sperm function, which involves the decrease of motility, viability,
and fertilizer capacity.
The quality of sperm in animals and human are typically assessed
through the measuring of sperm density, motility, total count, and
morphology [7]. Therefore, this work aimed to assess the stability of
the chromatin in ram sperm and relate it to the membrane integrity
structural – physiological and the rate of pregnancy.
MATERIALS AND METHODS
Study site
The handling and sampling of the animals were carried out at
the Experimental Station IVITA-El Mantaro, located 3300 meters
above sea level, in the Huancayo Province, Junin-Peru with a latitude
11°49'13"S and longitude 75°23'17"W. This station is developing
research to generate knowledge and new technologies based on
the livestock production systems. Its climate is characterized by an
annual average temperature of 12.4
o
C, annual average precipitation
of 707.9 millimeters (mm), and relative humidity of 77%.
Animals and semen collection
The present study was carried out using 5 male sheep (ram, about
120 kilograms -kg-) of 2 years old (YO) and 75 female sheep (ewes)
(about 85 kg) of 3-5 YO of the Junín sheep breed. During the day (d),
animals were kept daily grazed for 12 hours (h) on grass pastures of
Reed canarygrass (Phalaris arundinacea L.), and afternoons were fed
with alfalfa (Medicago sativa) hay to maintain a body condition. Each
ram was served with fteen ewes, generating ve (A, B, C, D, and E)
different Groups of analysis. For better control, rams and female sheep
belonging to the same Group were identied with their earrings and
colored wired laces. All rams were sexually active and under strict
control. For each ram, semen ejaculates were collected every month
(mos) for ve mos (a total of 25 semen samples) via an articial vagina
(robust tubular rubber (11 centimeter length) casing with rounded
opening, with a valve that allowed air to be added for adjustment of
inner pressure) and kept in a water bath (Thermo Scientic, Model
265, USA) at 30
o
C for 8-10 minutes (min) until examination of the initial
semen quality (after 16-18 h post sperm injection) for concentration,
sperm motility, volume, viability, and sperm plasma integrity through
eosin Y test and hypoosmotic (HOS) swelling test [26].
For this purpose, the split-plot design was applied. The samples
collected were transported to the Reproductive Biotechnology
laboratory of the National University of Center of Peru.
Seminal quality
Seminal volume, sperm morphology, sperm motility, and sperm
morphology were evaluated according to guidelines recommended
by World Health Organization (WHO) [30].
Sperm motility
Sperm motility was measured using a computer-assisted sperm
motion analyzer (Stromberg Mika, Germany). Semen was diluted
to a concentration of 30×10
6
sperm·millilitre
-1
(sperm·mL
-1
) with
a Buffer (Hepes balanced saline (HPS) at 7.6 pH) and then placed
approximately 10 microlitre (µL) on a 10 micrometer (µm) of depth
disposable chamber slide (Leja Products, Netherlands) [21]. The
image was digitized via a camera (10-phase contrast objective x and
3.3-ocular lens x) mounted on a phase-contrast microscope (x400)
(IM3-MET, OPTIKA, Italy). In total, ve small squares; one central and
four angulars were evaluated.
Sperm concentration
Sperm (spz) concentration was measured using a pre-calibrated
photometer (NucleoCounter SP100, ChemoMetec, Denmark) after
dilution 25 µL of ram semen with 500 µL of 0.9% NaCl solution
containing 0.32 millimolar (mM). Sperm concentration was computed
by counting cells in ve large squares of a Neubauer hemocytometer
(Countess 3 Automated Cell counter, Thermo Fisher Scientic, USA)
chamber under a phase contrast microscopy [21].
Eosin Y test and hypoosmotic swelling test
The structural integrity of the membrane of the sperm was
evaluated with the Eosin Y-water (EY) test method, and the functional
integrity was assessed through the hypoosmotic (HOS) swelling test
[21]. Results were expressed in percentage according to the degree
of reaction. It was considered a minimum count of 100 sperm per test.
•
Eosin Y – water method: here 0.5 grams (g) of eosin Y and
0.9 g of sodium chloride (NaCl) were dissolved in 100 mL of
distilled water. At the prepared eosin Y solution was added
10 g of nigrosine, and after, boiled, and allowed to cool at
room temperature. Then, the staining mixture was ltered
using lter paper and poured into a dark bottle. The tests
were performed using a drop of semen placed on a heated
microscope slide and added two drops of eosin-nigrosine
staining solution, and then mixture carefully using a pipette
tip for making a smear, and nally dried to room temperature
for 5 min. The smear was evaluated under a phase-contrast
microscope (Labomed Lx400, USA). At least 100 sperm were
assessed per slide, and the percentage of swollen tail sperm
was computed [20].
________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32157, 1 - 8
3 of 8
•
Hypoosmotic swelling test (HOS): ecacy of the HOS test was
evaluated using fructose (1351 milligrams -mg-), Na-citrate (735
mg), and 100 mL distilled water solution with three different
osmolarities: 50, 100, and 150 osmolarities (mOsm·kg
-1
). For
this, 0.1 mL ram semen was added to 0.9 mL of hypoosmotic
solutions (50 and 100 mOsm·kg
-1
fructose) and incubated
(Model 4130, Thermo Fisher Scientic, Brazil) at 37
o
C for 45
min at room temperature [19]. After incubation, one drop
of incubated semen was placed on a microscope slide and
assessed under phase contrasts x40 microscope. A minimum
of 100 sperm were assessed per slide, and percentages of
sperm with swollen were computed [19].
Sperm chromatin stability assessment
Sperm nuclear chromatin stability (NCS) was assessed following
the Gonzales and Sánchez [11] methodology. For this, 20 μL of fresh
semen were deposited in three test tubes coded from 1 to 3. In tube
1 (control) was added 180 μL of Borate Buffer (BB), in tube 2 was
added 180 μL of Sodium Dodecyl Sulfate (SDS) at 1%, and in tube 3
was added 180 μL of a mixture of Ethylenediaminetetraacetic acid
(EDTA) + SDS. These solutions were incubated (GTOP model 260, Olabo,
China) at 40
o
C for 60 min. Finally, 200 μL of glutaraldehyde at 2.5%
was added, mixed, and left at rest for 10 min. Each solution obtained
was smeared onto a glass slide, dried, xed with methanol (5 to 10
min), stained Giemsa solution (at 50%) for 40 min, and observed at the
microscope with x400 magnication. The minimum count was 100
sperm for each of the three laminae. Using this method, it is possible
to nd three grades: Grade 0: no condensation of head sperm head;
Grade 1: moderate condensation of head sperm; and Grade 2: high
condensation of the sperm head, all related to chromatin response
[9]. If the percentage of no-decondensed sperm head in the presence
of SDS + EDTA is greater-than 30%, the samples are classied as
having high sperm head stability [21].
Pregnancy rate related to the sperm chromatin stability
The pregnancy rate was evaluated using the birth rate, which was
calculated on the number of offspring born, to determine female
fertility.
Statistical analysis
The Wilcoxon rank-sum test was applied to compare the average
percentages of sperm concentration, seminal volume, normal
morphology, and sperm motility. Pearson correlation was computed
to assess the relationships among the eosin Y test, hypoosmotic test,
morphology, and motility. All data were treated with R-free software,
version 3.3.6 [24]. A comparison was considered signicant when
P was less-than 0.05.
RESULTS AND DISCUSSION
Seminal parameters quality
Average and standard deviation (SD) of sperm concentration,
seminal volume, normal sperm morphology, and sperm motility,
from sheep semen of ve Groups of rams are presented in TABLE
I. In that TABLE I has been observed differences (P<0.05) among
Groups for sperm concentration, but no differences were found for
seminal volume, normal morphology, and sperm motility. Higher
sperm concentration was presented by the ram belonging to Group
A, while the ram belonging to Group E showed the highest values
for seminal volume, normal morphology, and sperm motility. Sperm
motility ranged from 4.03 to 4.81 suggesting that the semen is of
good quality.
TABLE II has presented the percentages and SD of eosin swollen tail
sperm based on the Eosin Y test (structural integrity) and Hypoosmotic
test (physiological integrity). The percentage of sperm in Eosin Y
ranged from 58 to 77%, while the Hypoosmotic test ranged from
60 to 81%. The percentage average between methods no-showed
a signicant difference (P>0.05). Likewise, no differences (P>0.05)
were found between each Group. A higher percentage of sperm was
observed in Group E in both methods.
Regarding the structure and membrane physiology of sperm, these
variables are quite consistent in the contingency tables for membrane
structure, where eosin evidences a difference between cells that do
respond to the membrane test; while for the hypoosmotic test it was
more signicant because the sperm populations of the animals show
a greater difference between the swollen and non-swollen cells. Thus,
results found in ejaculated evidence to be optimal conditions for the
sperm populations to fulll their role of fertilizing the female gamete.
The reliability of the test results is supported by the correlation of
these in the measurement of an association with the same trend.
TABLE I
Seminal parameters from ejaculated ram semen
Group
Sperm concentration
sperm
(10
6
·mL
-1
) (x̄ ± SD)
Seminal volume
(mL) (x̄ ± SD)
Normal morphology
(%) (x̄ ± SD)
Sperm motility
(x̄ ± SD)
A
2304 ± 386 a 0.96 ± 0.41 a 84 ± 7 a 4.61 ± 0.54 a
B
2198 ± 220 b 1.12 ± 0.22 a 70 ± 11 b 4.63 ± 0.55 a
C
2110 ± 196 b 0.90 ± 0.33 a 89 ± 4 a 4.22 ± 0.83 a
D
2228 ± 140 c 0.88 ± 0.34 a 91 ± 4 a 4.03 ± 1.02 a
E
2090 ± 263 b 1.56 ± 1.31 a 92 ± 5 a 4.81 ± 0.45 a
Total
2186 ± 246 1.08 ± 0.68 85 ± 11 a 4.44 ± 0.71
n= 5: number of ejaculations, (x̄ ± SD): average and standard deviation, values on each vertical column followed
by the same letter do not dier signicantly at
P<0.05
Sperm chromatin stability in Sheep of the Junín race/ Unchupaico-Payano et al. ______________________________________________________
4 of 8
Pearson correlation between observed parameters found in ram
semen was presented in TABLE III. There were no signicant correlations
between motility and the Eosin Y test and the Hypoosmotic test
(P<0.001), except for morphology. A signicant correlation (r
2
=0.81) was
observed between the Eosin Y and Hypoosmotic test (P<0.001). Besides,
morphology and Hypoosmotic tests showed statistical correlation.
Morphological and kinetic aspects of sperm are important factors
for fertilization [10]. For this, microscopic analysis is necessary to
contrast the mass and individual motility of sperm the most before
possible. Thus, to avoid thermal shock, a platen adaptable to the base
of the microscope was used.
Osmotic pressure is of great importance, not in the ejaculate itself
whose variations do not exceed normal limits, but in terms of the
composition of the diluents [6]. In general terms, it can be admitted
that sperm requires for its normal maintenance isotonic means [15].
Variations in the osmotic pressure of solvents or culture media
are not compatible with sperm vitality. However, different slightly
hypotonic means have been tested, with the idea that this means
greater possibilities of natural detoxication of the cell spermatic,
a phenomenon that would favor the survival and the fecundating
capacity of the sperm. It seems that ruminant sperm are the most
resistant to the osmotic pressure, while the gametes of animals
of internal fertilization require for their normal biology very few
variations of the same. In this case, the hypoosmotic test was used
to test the functional eciency of the sperm membrane, which gives
an acceptable percentage in the sperm population subjected to
hypoosmotic conditions, indicating an adequate fertile potential, with
an optimal functionality of their sperm membrane. High percentages
of sperm cells that were subjected to the hypoosmotic test suggest
that sperm populations subjected to membrane integrity tests (eosin
and hypoosmotic testing) are crucial for viability and physiological
changes that occur on the membrane surface [18], so it can be argued
that both tests would have a value in testing sperm fertility.
Membrane integrity is important for sperm metabolism, training,
acrosomal reaction, and sperm binding to the surface of the oocyte
[4]. In contrast, damage to membrane integrity can cause loss of
normal sperm function, such as motility, viability, and fertilizing
capacity. It has been shown that exposure of mammalian sperm to
hypoosmotic conditions can cause water ow through the membrane,
resulting in increased sperm volume and swelling of the plasma
membrane. The ability of the sperm tail to swell in the presence of a
hypoosmotic solution is a sign that the transport of water through the
membrane occurs normally. Swelling of the spermatic nucleus and
decondensation of chromatin in mammalian sperm occurs shortly
after penetration of the sperm to the cytoplasm of the oocyte. The
decondensation of chromatin was doubled when the pre-ejaculates
of ram and cattle were incubated in vitro. There was an increase
in the time of decondensation when stored in vitro at 25 °C, which
reected an increase in the junctions within the sperm by histones
for the formation of disulde bridges. This can increase embryonic
mortality when it is observed that sperm was stored in vitro before
insemination [16].
Chromatin compaction degrees
Interactions between animals (Groups A to E), methods (BB, SDS,
and SBS + EDTA), months, and compaction levels are presented in
TABLE IV. From TABLE IV, it was observed no signicant (P>0.05)
differences among methods. No differences may be related to
the that the medium used would depend on the degree that has
each ejaculate. However, compaction degrees were observed with
signicant differences (P<0.05) when the methods interacted with the
periods or mos in which the semen was collected. In the case of mos
that interact with the degrees of compaction was found signicant
differences (P<0.001). Interaction mos*compaction degrees being
signicant ensures that each mos evaluated maintains different
conditions relating to the compaction degree, and suggests that any
animal may be evaluated. Likewise, it was found that the compaction
degree was affected (P<0.0001), by the interaction (mos*compaction
degree, (P<0.001)) with the mos in which the ejaculation was obtained.
Besides, was observed interactions between the compaction degrees
with the methods (P<0.001) and mos (P<0.01) evaluated. Unlike the
degrees of compaction, no simple effect was found in the animals or
the mos that the semen was collected. However, differences between
the methods were signicant when interacting with the mos and the
degrees of compaction.
TABLE II
Percentages and standard deviation of eosin swollen tail
spermatozoa based on Eosin Y test (structural integrity)
and Hypoosmotic test (physiological integrity)
Group
Eosin Y test
100 sperm (%)
Hypoosmotic test
100 sperm (%)
A
68 ± 15 a
60 ± 12 a
B
58 ± 8 a
61 ± 7 a
C
72 ± 4.5 a
75 ± 12 a
D
72 ± 26 a
68 ± 32 a
E
77 ± 11 a
81 ± 8 a
Average
69.4 ± 13 A
69.0 ± 14 A
n= 5: number of ejaculations, (x̄ ± SD): average and standard deviation
TABLE III
Pearson correlation between observed parameters of ram semen
Observed semen
parameters
Eosin Y test (%) Hypoosmotic test (%) Morphology (%) Motility (0-5)
Eosin Y test (%)
1
Hypoosmotic test (%)
0.81 (
P<0.001) 1
Morphology (%)
0.56 (
P<0.001) 0.46 (P=0.001*) 1
Motility (0-5)
0.22 (P<0.05) 0.22 (P=0.002*) 0.52 (P<0.001) 1
*: signicative value
________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32157, 1 - 8
5 of 8
Differences in compaction degrees found among the males studied
showed dependence on the effect of the period in which the ejaculates
were obtained (compaction interaction*mos: significant). This
result suggests the presence of the seasonal effect in the process
of spermatogenesis on the compaction of nuclear chromatin [25].
However, rams are not mostly susceptible to the effect of seasonality
[29]. This was corroborated with results found in seminal parameters
quality (concentration, normal morphology, and sperm motility) which
were not altered. However, it was observed an effect on the compaction
degrees of the chromatin.
In the interaction with methods, the highest percentage occurred
between grades 0 and 1, which expresses appropriate stability of
chromatin, good quality of the sperm, and excellent fecundation
potential [14]. In the present case, the BB method reported a higher
percentage of sperm compared to other methods, with a grade of
0 in all rams. This could be because SDS is a detergent that acts on
the disulde bridges of chromatin, which makes sperm chromatin
less stable when subjected to SDS detergent. As the EDTA is a tinging
agent that acts on zinc by removing it; this mineral gives stability to
the disulde bridges of sperm chromatin [3]. This nding suggests
a line of good quality that guarantees their fertilizer capacity.
TABLE IV
Split plot analysis showing interaction levels between animals,
methods, months, and compaction degrees
Source DF Type SS Square F - value P
Animals 4 7.11 1.78 0.02 0.999
Month 4 4.89 1.22 0.01 0.999
Error (a) 16 12.89 0.806 0.01 1.000
Month 2 8820.2 4410.1 46.65 0.0001
Compaction*compaction 8 2519.8 314.9 3.33 0.0018
Error (b) 40 17177 429.42 4.54 0.0001
Methods 2 1.56 0.78 0.01 0.9918
Method*month 8 8.44 1.06 0.01 1.0000
Method*compaction 4 27587.8 6896.9 72.96 0.0001
Method*month*compaction 16 2808.9 175.6 1.86 0.0312
Error (c) 120 11343.3 94.53
Total 224 70291.6
TABLE V showed the physiological state and compaction degree of
sperm chromatin. From TABLE V, there were no differences between
pregnancy and non-pregnancy rates. There is also no statistical
relationship (P>0.05) between compaction degrees (0, 1, and 2) and
their relationship with animals that were not as prey. However, a
higher rate of prey (74.6%) was found.
In the pregnancy rate and its relationship with the stability of
chromatin, it was found a non-signicant correlation, which would
suggest that there is no adequate degree for the good stability of
chromatin, which did not coincide with being the most frequent in
pregnancy. For this, standard semen analysis is required, including
concentration, morphology, and motility analysis, which are widely used
as an indicator of fertility [13]. Thus, the parameters above mentioned
may inuence the physiological, structural, and stability aspects of
the sperm chromatin. Based on all that, here the method used allowed
obtain sperm of the best quality (in terms of morphology and motility),
with minimum contamination by other cellular or non-cellular structures
such as granulations and crystals [27].
Ejaculates of Ovid’s and caprids, as well as that of ruminants in
general, are composed of four fractions or liquid emissions, the product
of the litter glands, bulbourethral, prostate, Henle blisters, and vesicular
glands [5]. The largest volume corresponds to the prostate glands and
blisters of Henle; hence, the function of this biological phenomenon.
Here, it is possible to admit that the ejaculate of sheep (Ovies aries)
and goats (Capra aegagrus hircus) is of low volume and high sperm
concentration since in all species the volume of the ejaculate depends
on the secretory capacity of the gland’s paragenitals.
Pregnancy rate related to the sperm chromatin stability
TABLE VI presents the number of sheep by group sent for
reproduction, served, calved, and served percentage. From TABLE
VI, Group B (93.30%) showed a higher percentage of sheep served,
TABLE V
Physiological state and compaction degree of sperm chromatin
Physiological
state
Compaction degrees
Total
0 1 2
(< 0.63) (0.63 to 1.17) (> 1.18)
n % n % n % n %
No prey
6 24.0 5 20.0 8 32.0 19 25.3
Prey
19 76.0 20 80.0 17 68.0 56 74.7
Total
25 25 25 75 100
Sperm chromatin stability in Sheep of the Junín race/ Unchupaico-Payano et al. ______________________________________________________
6 of 8
followed by Group C and D (both with 86.67%), and Group A and E (both
with 80.0%). About calved sheep, percentage of prolicity, and sex of
the offspring, Group B obtained the highest number of calved sheep
(13), followed by Group C and D (both 12), and Group A and E (both
10). The average percentage of prolicity was 114%, Group B (123%)
presented the highest percentage, followed by Group D (118%), Group
C (116%), Group A (110%), and Group E (100%). Regarding Pearson’s
correlation between pregnancy rate and sperm chromatin stability,
it was found a negative correlation (-0.11) indicating that there is
no statistical signicance (P>0.05). It is possible to suggest that
the higher the degree of de-condensation of chromatin the rate of
calving remains. The dispersion of the degrees of compaction tends
to increase the percentage of calving, thus, the point “66.67” (66.67,
0.99), the point “80” (80, 0.93), and the point “86.67” (86.67, 0.89), from
which it follows that by increasing the percentage of calves tends to
decrease the degree of compaction, from 0.99 to 0.82.
The results obtained regarding the timely decondensation of
chromatin when incubated in BB as a primary and common mean
at three degrees of condensation at a temperature of 40 °C for
approximately 45 min, in no way resemble being stored for a few h
or d, indicating the importance of temperature variation is for the
case of articial insemination (AI) programs and the considerations
that must be given for obtaining good results, that is, high percentages
of pregnancy and viability of embryos. During the late stage of
spermiogenesis, the nucleus of the spermatid becomes very condensed
and adopts an elongated shape characteristic of the species. Although
there is no full understanding of the forces that determine nucleus
shape, it is known that these visible modications are accompanied
by biochemical changes involving the removal of Ribonucleic acid
(RNA) from the nucleus at the beginning of nuclear elongation and
the replacement of lysine-rich histone with a more basic arginine-
rich protein.
Sperm obtained from sheep healthy were induced to decondensation
of chromatin with dithiothreitol (DTT) and SDS in vitro. A high range
of stable no-condensed nuclei (79%) was observed in ram semen
with normal fertility. In contrast, semen of rams with low fertility
presented 31% of chromatin stability. Seminal plasma and other
constituents (Zn) inhibited the decondensation chromatin of sperms,
while when added EDTA shows the opposite. Results suggest that the
addition of chromatin decondensation using SDS under controlled
conditions may be a reliable method for predicting fertile capacity
[23]. The results also showed the EDTA action to eliminate Zn, and
borate buffer, allowing their growing inside the sperm medium
[17]. Likewise, it was observed that there was no inhibition of the
decondensation chromatin as happened in the tube containing SDS
and BB. It is important to mention that the methodologies evaluated
in this work are different, due here applying the protamine P2, which
is held by the bull, ram, rat, pig, guinea pig, and humans [12].
This makes chromatin less stable in humans than in other species
[7]. These results coincide with the obtained results. In this sense,
the sperm subjected to the EDTA does not have a very decondensed
form, which does not mean that there has not been decondensation.
This is probably by the minimum presence of protamine P1, which
makes the chromatin of sheep more stable.
These results offer the idea that the condensation of chromatin in
mammals tends to vary because of the pregnancy rate. In this study,
the pregnancy rate in Junín sheep was 86.67%.
Although it is true not there is a degree of condensation that differs
in function from the fertilization rate, which is explained by the number
of sheep pregnant and not pregnant. The degrees of condensation of
chromatins 1 and 2 are the least stable, compared to the zero degrees
which are the most stable [22].
TABLE VII shows the rate of fertilization of each Group based on
the pregnancy and not the pregnancy of the sheep. No signicant
differences (P<0.05) were found between the animal Group. These
results indicate that none of the rams was more effective in
reproduction than the others.
CONCLUSIONS
The concentration of sperm showed differences among the ve
groups analyzed. Based on the parameters assessed, the seminal
volume and sperm motility do not show signicant differences among
groups. Besides, a high correlation (r
2
=0.52) was found between
motility and morphology. A high rate of fertilization rate (74.6%.) was
TABLE VI
Number of sheep by group sent for reproduction, served,
calved, and served percentage
Group
sheep for
reproduction
Sheep
served
Calved
sheep
Served
percentage (%)
A
15 12 10 80.00
B
15 14 13 93.30
C
15 13 12 86.67
D
15 13 11 86.67
E
15 12 10 80.00
Total
75 64 60
TABLE VII
Rate of fertilization from the ram that pregnant and did not pregnant the sheep
Fertilization Statistics
Group of animals
Total
A B C D E
Prey
Recount
(% of total)
10
(13.3%)
13
(17.3%)
12
(16.0%)
11
(14.7%)
10
(13.3%)
56
(74.7%)
No prey
Recount
(% of total)
5
(6.7%)
13
(17.3%)
12
(16.0%)
11
(14.7%)
10
(13.3%)
56
(74.7%)
Total
Recount
(% of total)
15
(20.0%)
15
(20.0%)
15
(20.0%)
15
(20.0%)
15
(20.0%)
56
(74.7%)
________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32157, 1 - 8
7 of 8
found. It was concluded in general that techniques to evaluate nuclear
condensation values do have a high likelihood to give a diagnosis about
the future potential of sperm populations in Junín ram.
ACKNOWLEDGEMENTS
Authors thanks the faculty of Veterinary Medicine of the Universidad
Nacional Mayor de San Marcos (UNMSM) and the Experimental
Station IVITA-El Mantaro, for their collaboration in the logistic and
developments of this research.
BIBLIOGRAPHIC REFERENCES
[1] BALHORN, R.; STEGER, K.; BERGMANN, M.; SCHUPPE, H.C.;
NEUHAUSER, S.; BALHORN, M.C. New Monoclonal Antibodies
Specific for Mammalian Protamines P1 and P2. Syst. Biol.
Reprod. Med. 4(6): 424–447. 2018. https://doi.org/jctc.
[2] BINDARI, Y.R.; SHRESTHA, S.; SHRESTHA, N.; GAIRE, T.N. Effects
of Nutrition on Reproduction- A Review. Pelagia Res. Libr. 4(1):
421–429. 2013.
[3] BJÖRNDAHL, L.; KVIST, U. Human Sperm Chromatin Stabilization:
A Proposed Model Including Zinc Bridges. Mol. Hum. Reprod.
16(1): 23–29. 2009. https://doi.org/djrrvx.
[4] BUCCI, D.; SPINACI, M.; GALEATI, G.; TAMANINI, C. Different
Approaches for Assessing Sperm Function. Anim. Reprod. 16(1):
72–80. 2018. https://doi.org/jctd.
[5] CARVALHO, L.E.; SILVA-FILHO, J.M.; PALHARES, M.S.; SALES,
A.L.R.; GONCZAROWSKA, A.T.; OLIVEIRA, H.N.; RESENDE, M.;
ROSSI, R. Physical and Morphological Characteristics of the
First Three Jets of Pêga Jackasses Sperm-Rich Fraction. Arq.
Bras. Med. Vet. Zoot. 68(4): 845–852. 2016. https://doi.org/jctf.
[6] COOPER, T.G. The Epididymis, Cytoplasmic Droplets and Male
Fertility. Asian J. Androl. 13(1): 130–138. 2011. https://doi.org/
dfv5ww.
[7] EVENSON, D.; JOST, L. Sperm Chromatin Structure Assay Is
Useful for Fertility Assessment. Springer. 22(2): 169–189. 2000.
[8] GALIOTO, F.; PAFFARINI, C.; CHIORRI, M.; TORQUATI, B.;
CECCHINI, L. Economic, Environmental, and Animal Welfare
Performance on Livestock Farms: Conceptual Model and
Application to Some Case Studies in Italy. Sustain. 9(9): 1–22.
2017. https://doi.org/gb4rb6.
[9] GARCÍA, J.; NORIEGA-HOCES, L.; GONZALES, G.F. Sperm
Chromatin Stability and Its Relationship with Fertilization Rate
after Intracytoplasmic Sperm Injection (ICSI) in an Assisted
Reproduction Program. J. Assist. Reprod. Genet. 24(12):
587–593. 2007. https://doi.org/d3v9pf.
[10] GARCÍA-VÁZQUEZ, F.; GADEA, J.; MATÁS, C.; HOLT, W. Importance
of Sperm Morphology during Sperm Transport and Fertilization
in Mammals. Asian J. Androl. 18(6): 844–850. 2016. https://doi.
org/jctg.
[11] GONZALES, G.F.; SÁNCHEZ, A. High Sperm Chromatin Stability
in Semen with High Viscosity. Syst. Biol. Reprod. Med. 32(1):
31–35. 1994. https://doi.org/bkbvpb.
[12] GU, N.H.; ZHAO, W.L.; WANG, G.S.; SUN, F. Comparative Analysis of
Mammalian Sperm Ultrastructure Reveals Relationships between
Sperm Morphology, Mitochondrial Functions and Motility. Reprod.
Biol. Endocrinol. 17(1): 1–12. 2019. https://doi.org/jcth.
[13] HAMILTON, J.A.M.; CISSEN, M.; BRANDES, M.; SMEENK, J.M.J.;
DE BRUIN, J.P.; KREMER, J.A.M.; NELEN, W.L.D.M.; HAMILTON,
C.J.C.M. Total Motile Sperm Count: A Better Indicator for the
Severity of Male Factor Infertility than the WHO Sperm Classication
System. Hum. Reprod. 30(5): 1110–1121. 2015. https://doi.org/f7c9q8.
[14] HEKMATDOOST, A.; LAKPOUR, N.; SADEGHI, M.R. Sperm
Chromatin Integrity: Etiologies and Mechanisms of Abnormality,
Assays, Clinical Importance, Preventing and Repairing Damage.
Avicenna J. Med. Biotechnol. 1(3): 147–160. 2009.
[15] HOLMES, E.; BJÖRNDAHL, L.; KVIST, U. Hypotonic Challenge
Reduces Human Sperm Motility through Coiling and Folding of
the Tail. Androl. 52(11): 1–7. 2020. https://doi.org/hxjr.
[16] KURYKIN, J.; HALLAP, T.; JALAKAS, M.; PADRIK, P.; KAART, T.;
JOHANNISSON, A.; JAAKMA, Ü. Effect of Insemination-Related
Factors on Pregnancy Rate Using Sexed Semen in Holstein Heifers.
Czech J. Anim. Sci. 61(12): 568–577. 2016. https://doi.org/f9r8jm.
[17] MAARES, M.; HAASE, H. A Guide to Human Zinc Absorption:
General Overview and Recent Advances of in Vitro Intestinal
Models. Nutrients. 12(3): 1–45. 2020. https://doi.org/gqfrs7.
[18] MULLER, C.J.C.; CLOETE, S.W.P.; BOTHA, J.A. Fertility in Dairy
Cows and Ways to Improve It. S. Afr. J. Anim. Sci. 48(5): 858–868.
2018. https://doi.org/jctj.
[19] MUNUCE, M.J.; CAILLE, A.M.; BERTA, C.L.; PERFUMO, P.;
MORISOLI, L. Does the Hypoosmotic Swelling Test Predict
Human Sperm Viability? Arch. Androl. 44(3): 207–212. 2000.
https://doi.org/d8bx6g.
[20] NUR, Z.; SEVEN-CAKMAK, S.; USTUNER, B.; CAKMAK, I.; ERTURK,
M.; ABRAMSON, C.I.; SAĞIRKAYA, H.; SOYLU, M.K. The Use of
the Hypo-Osmotic Swelling Test, Water Test, and Supravital
Staining in the Evaluation of Drone Sperm. Apidologie. 43(1):
31–38. 2012. https://doi.org/bkt786.
[21] RAMÓN, M.; SALCES-ORTIZ, J.; GONZÁLEZ, C.; PÉREZ-GUZMÁN,
M.D.; GARDE, J.J.; GARCÍA-ÁLVAREZ, O.; MAROTO-MORALES, A.;
CALVO, J.H.; SERRANO, M.M. Inuence of the Temperature and the
Genotype of the HSP90AA1 Gene over Sperm Chromatin Stability
in Manchega Rams. PLoS One. 9(1): 1–9. 2014. https://doi.org/jctk.
[22] RIBAS-MAYNOU, J.; GARCIA-BONILLA, E.; HIDALGO, C.O.;
CATALÁN, J.; MIRÓ, J.; YESTE, M. Species-Specic Differences
in Sperm Chromatin Decondensation Between Eutherian
Mammals Underlie Distinct Lysis Requirements. Front. Cell
Dev. Biol. 9(4): 1–11. 2021. https://doi.org/jctm.
[23] RODRIGUEZ, H.; OHANIAN, C.; BUSTOS‐OBREGON, E. Nuclear
Chromatin Decondensation of Spermatozoa in vitro: A Method for
Evaluating the Fertilizing Ability of Ovine Semen. Int. J. Androl.
8(2): 147–158. 1985. https://doi.org/cnt9dq.
[24] R TEAM CORE. A Language and Environment for Statistical
Computing. R Foundation for Statistical Computing, Vienna
Austria. R Foundation for Statistical Computing: Vienna, Austria.
Version 3.6.2. Pp 3879. 2019.
Sperm chromatin stability in Sheep of the Junín race/ Unchupaico-Payano et al. ______________________________________________________
8 of 8
[25] SILVA, S.F.M.; OLIVEIRA, L.C.A.; DIAS, F.C.R.; CORDERO-SCHMIDT,
E.; VARGAS-MENA, J.C.; SILVA, I.G.M.; BÁO, S.N.; LUNA, J.L.S.; LIMA,
R.R.M.; JÚNIOR, R.F.A.; FARIAS, N.B.S.; MOURA, C.E.B.; MATTA,
S.L.P.; MORAIS, D.B. Seasonal Evaluation of Spermatogenesis
of the Hematophagous Bat Desmodus Rotundus in the Caatinga
Biome. PLoS One. 15(12): 1–19. 2020. https://doi.org/jctn.
[26] TAYLOR, M.A.; GUZMÁN-NOVOA, E.; MORFIN, N.; BUHR, M.M.
Improving Viability of Cryopreserved Honey Bee (Apis Mellifera
L.) Sperm with Selected Diluents, Cryoprotectants, and Semen
Dilution Ratios. Theriogenol. 72(2): 149–159. 2009.
[27] UGUR, M.R.; SABER ABDELRAHMAN, A.; EVANS, H.C.; GILMORE,
A.A.; HITIT, M.; ARIFIANTINI, R.I.; PURWANTARA, B.; KAYA, A.;
MEMILI, E. Advances in Cryopreservation of Bull Sperm. Front.
Vet. Sci., 6(6): 1–15. 2019. https://doi.org/gg8vj5.
[28] WATHES, D.C.; OGUEJIOFOR, C.F.; THOMAS, C.; CHENG, Z.
Importance of Viral Disease in Dairy Cow Fertility. Engineering.
6(1): 26–33. 2020. https://doi.org/jctp.
[29] WETTERE, W.H.E.V.; KIND, K.L.; GATFORD, K.L.; SWINBOURNE,
A.M.; LEU, S.T.; HAYMAN, P.T.; KELLY, J.M.; WEAVER, A.C.;
KLEEMANN, D.O.; WALKER, S.K. Review of the Impact of Heat
Stress on Reproductive Performance of Sheep. J. Anim. Sci.
Biotechnol. 12(1): 1–18. 2021. https://doi.org/jctq.
[30] WOLD HEALTH ORGANIZATION (WHO). WHO Laboratory Manual
for the Examination and Processing of Human Semen, 5
th
. Ed.;
World Health Organization. Pp 271. 2010.
[31] WU, T.F.; CHU, D.S. Sperm Chromatin: Fertile Grounds for
Proteomic Discovery of Clinical Tools. Mol. Cell. Proteomics.
7(10): 1876–1886. 2008. https://doi.org/dt79p3.
[32] ZHENG, W.W.; SONG, G.; WANG, Q.L.; LIU, S.W.; ZHU, X.L.; DENG,
S.M.; ZHONG, A.; TAN, Y.M.; TAN, Y. Sperm DNA Damage Has a
Negative Effect on Early Embryonic Development Following in
Vitro Fertilization. Asian J. Androl. 20(7): 75–79. 2018. https://
doi.org/gbkh4x.
