REPRODUCTIVE TRAITS AND FREEZABILITY OF SEMEN OF RAMS … · REPRODUCTIVE TRAITS AND FREEZABILITY...
Transcript of REPRODUCTIVE TRAITS AND FREEZABILITY OF SEMEN OF RAMS … · REPRODUCTIVE TRAITS AND FREEZABILITY...
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REPRODUCTIVE TRAITS AND FREEZABILITY OF SEMEN OF RAMS FROM TWO DESERT SHEEP
ECOTYPES IN THE SUDAN.
BY
Babiker Abdel Atti Elsharif
B.V.Sc., M.V.Sc. University of Khartoum.
A Thesis
Submitted in fulfillment of the requirements of
The University of Khartoum for the Degree of Ph.D.
Supervised by
Dr. Sharaf A. Makawi
B.V.Sc., Ph.D. University of Khartoum.
Department of Reproduction and Obstetrics,
Faculty of Veterinary Medicine.
January, 2010.
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Dedications To my father …………….....
To my mother……………..
To my wife……………..…
To my sons and daughter
With endless love
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Table of Contents
Dedication .............................................................................................................. II
Table of contents ................................................................................................. III
List of tables ...................................................................................................... VIII
List of figures.......................................................................................................IX
Acknowledgement. ............................................................................................ X
English Abstract ............................................................................................... XII
Arabic Abstract ................................................................................................ XV
Introduction ...................................................................................................XV1I
Chapter one: .............................................................................................
Review of literature: ..............................................................................1
1-Reproduction in sheep...........................................................................2
1-1 Libido and mating behaviour in rams ...............................................2
1-2 Semen . ..............................................................................................5
1-3 Semen characteristics. .......................................................................6
1-3-1 Colour and consistency of semen. ................................................7
1-3-2 Volume of ejaculate. ......................................................................8
1-3-3 Concentration of spermatozoa .......................................................8
1-3-4 Sperm motility. ..............................................................................9
1-3-5 Sperm cell morphology .................................................................9
1-4 Factors affecting semen production. .............................................11
1-4-1 Age of the ram. ............................................................................11
1-4-2 Nutrition. ......................................................................................11
1-4-3 Testes size. ...................................................................................14
IV
1-4-4 Environment ................................................................................17
1-4-4-1 Temperature. ............................................................................18
1-4-4-2 Photoperiod ..............................................................................19
1-4-5 Frequency of semen collection. ...................................................20
1-4-6 Breed ............................................................................................21
1-5 Seasonal effect on reproduction ......................................................22
1-6 Semen collection. ............................................................................24
1-6-1 Collection from the vagina after mating ......................................24
1-6-2Artificial vagina. ...........................................................................24
1-6-3 Electro-ejaculation. ......................................................................25
1-7 Semen dilution. ...............................................................................26
1-8 Semen freezing ................................................................................32
1-9 Cryopreservation of spermatozoa. ..................................................33
1-10 Thawing of frozen semen . ..........................................................35
1-11 Oestrous synchronization. .............................................................36
1-12 Insemination of ewes ....................................................................40
1-12-1 Vaginal insemination. ................................................................42
1-12-2 Cervical insemination. ..............................................................42
1-12-3 Laparoscopic insemination. ......................................................45
1-13 Fertility following AI. ...................................................................46
Chapter two: ............................................................................................
2- Materials and methods: ..................................................................51
2-1 Site of study. ...................................................................................51
2-2 Latitude and climate. .......................................................................51
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2-3 Animals. ..........................................................................................51
2-3-1 Housing ........................................................................................52
2-3-2 Feeding . ......................................................................................52
2-3-3 Body weight recording. ...............................................................53
2-3-4 Scrotal circumference measurements. .........................................53
2-4 Reaction time. .................................................................................53
2-5 Semen collection. ............................................................................53
2-6 Semen evaluation ............................................................................55
2-6-1 Appearance of ejaculate. ............................................................55
2-6-2 Volume of ejaculate ....................................................................55
2-6-3 Colour and consistency of semen. ..............................................55
2-6-4 Sperm motility. ...........................................................................56
2-6-4-1 Mass activity . ..........................................................................56
2-6-4-2 Individual motility. ...................................................................57
2-6-5 Sperm cell concentration . ..........................................................57
2-6-6 Sperm cell morphology. .............................................................59
2-6-6-1 Wet preparation. .......................................................................59
2-6-6-2 Dry preparation ........................................................................59
2-6-7 Live-dead spermatozoa. .............................................................59
2-7 Extender preparation. ...................................................................60
2-8 Semen dilution. .............................................................................60
2-9 Semen cooling. ...............................................................................61
2-10 Semen packing. .............................................................................61
2-11 Semen freezing. .............................................................................62
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2.12 Pre- and post-freezing rejected samples ………………………....62
2-13 Thawing of frozen semen. ...........................................................62
2-14 Oestrous synchronization. .............................................................62
2-15 Insemination of ewes. ..................................................................63
2-16 Statistical analysis .........................................................................66
2-17 Conception rate…………………………………………………..64
2-18 Experimental design……………………………………………..64
2-19 Statistical analysis………………………………………………..65
Chapter three ...........................................................................................
3-Results: ..............................................................................................68
3-1 Semen characteristics ......................................................................68
3-1-1 Colour and consistency of ejaculate. ...........................................68
3-1-2 Ejaculate volume. ........................................................................69
3-1-3 Mass activity. ...............................................................................70
3-1-4 Individual motility .......................................................................71
3-1-5 Sperm cell concentration. ............................................................72
3-1-6 live sperm count. .........................................................................72
3-1-7 Sperm cell abnormalities. ...........................................................73
3-2 Live body weight. ........................................................................74
3-3 Scrotal circumference. .................................................................75
3-4 Relationship between body weight, scrotal circumference, semen
volume and sperm cell concentration.....................................................76
3.5 Reaction time. ................................................................................77
3-6 Freezability of semen. ..................................................................79
VII
3-7 Conception rate. ...........................................................................81
Chapter four: ...........................................................................................
Discussion: ............................................................................................84
4-1 Semen characteristics. .....................................................................85
4-1-1 Colour and consistency of ejaculate ............................................85
4-1-2 Ejaculate volume .........................................................................85
4-1-3 Mass activity. ...............................................................................87
4-1-4 Individual motility. ......................................................................87
4-1-5 Sperm cell concentration. ............................................................88
4-1-6 Live Sperm count. ........................................................................89
4-1-7 Sperm cell abnormalities. ............................................................90
4-2 Scrotal circumference. ....................................................................92
4-3 Reaction time. .................................................................................93
4-4 Freezability of spermatozoa. ...........................................................94
4-5 Rejection rate. ..................................................................................95
4-6 Fertility following AI. .....................................................................96
4-7 Conclusions and recommendations. ...............................................98
5- References. .....................................................................................100
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List of Tables
1-............................................................................................... N
umber and percentage of ejaculates with different colours and
consistency of Hamari and Kabashi rams..................................68
2- Number and percentage of watery ejaculates in different seasons
of the year of Hamari and Kabashi rams………………………69
3- Means and standard errors of semen traits in different breed….70
4- Means and standard errors of semen traits in different seasons.71
5- Means and standard errors of sperm cell abnormalities in
different breed………………………………………………….74
6- Means and standard errors of sperm cell abnormalities in
different seasons……………………………………………….75
7-............................................................................................... E
ffect of season and breed on scrotal circumference and body
weight..........................................................................................79
8-............................................................................................... P
erson`s correlation coefficient between body weight, scrotal
circumference, semen volume and sperm cell concentration from
Hamari rams................................................................................80
9-............................................................................................... P
erson`s correlation coefficient between body weight, scrotal
circumference, semen volume and sperm cell concentration from
Kabashi rams...............................................................................80
10‐The reaction time of Hamari and Kabashi rams in different
seasons of the years ....................................................................81
11-The effect of breed and season on pre- and post-freezing rejection
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batches and post-thaw motility of frozen semen……………...…82
12-Fertility parameters of frozen-thawed semen obtained from
Hamari and Kabashi rams ........................................................82
List of figures
1- The effect of season on sperm abnormalities of Hamari rams…………………………………………………………….76
2- The effect of season on sperm abnormalities of Kabashi rams…………………………………………………………….77
3- The effect of season on pre- and post-rejection sample rate of Hamari rams…………………………………………….76
4- The effect of season on pre- and post-rejection sample rate of Kabashi rams………………………...………………….76
5- The effect of breed on pre-, post and total percentage of rejected samples ……………………………………………….76
Appendix:
1-Climatic conditions in Khartoum during the experimental period..141
2-Semen characteristics Anova table…………………..…………….142
3-Sperm cell abnormalities Anova table…………………..…………143
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Acknowledgements
I wish to express my sincere gratitude to Dr. Sharaf A. Makawi,
Department of Reproduction and Obstetrics, Faculty of Veterinary
Medicine, University of Khartoum, for his keen supervision, deep
interest, invaluable advice and guidance during this work, to whom I am
very grateful.
My sincere acknowledgement is also due to Dr. Faisal Hassan
Ibrahim, the former executive manager of LADCO (Live-stock and
Agricultural Development Company) whose financial support,
enthusiasm and close co-operation have made this investigation
possible.
I am most grateful to Dr. Adil Salim Al Garai, Faculty of
Veterinary Medicine, University of Khartoum, Dr. Khalid Alssabayil,
King Saud University, Qasim, Agriculture and Veterinary Collage,
K.S.A., Mr. Mohamed Zein Elsharif, Dr.Suhair Sayed, for their fruitful
assistance, for providing me with many scientific papers.
For expert technical assistance I wish to acknowledge my
indebtedness to my colleagues at the National Animal Breeding and AI
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Centre, Kuku, Dr. Mohamed Saad, Dr. Salih Graham, Miss. Somaya
Mohamed Adam and Miss. Husna Hussain.
I would like to express my sincere gratitude to Mr. Taha Yasin,
for his assistance for the application of the oestrous synchronization and
AI programme in ewes, and I am most grateful for his friendship.
My thanks are also due to Mr. Emam, Mr. Abdalla Dago and
Mr. Abu Amna Osman, for their care after the animals during the course
of this study.
Also I wish to thank Dr. Mohamed Khair Abdella, Faculty of
Animal Production, University of Khartoum, for his help with statistical
analysis and results of this experiment.
My thanks also due to Meteorology Department and Mahmoud
El Hassan, for providing me with meteorological data.
Last, but not the least, I wish to sincerely thank my family, my
wife, my sons, Ayman, Ahmed, Mohanad and Awap and my daughter
Shemookh. They know how to create a comfortable studying
environment for me.
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Abstract
The objective of this experiment was to study the influence of breed
and season on sexual performance and semen characteristics of two
Sudan Desert sheep ecotypes. Freezability and fertility of frozen semen,
collected from the two breeds, was also investigated. The rams were
2 - 4 years old at the start of the experiment with initial average live
body weights of 73.60 and 61.20 kg for Hamari and Kabashi rams,
respectively. All animals were maintained under uniform conditions of
feeding and management. A total of 293 semen ejaculates were
collected by means of an artificial vagina from six Hamari and three
Kabashi rams. Data of Hamari and Kabashi rams showed that the over-
all means for seminal attributes were 1.31 and 1.23 ml for semen
volume, 2.73 and 2.37 scores for mass motility, 68.11% and 67.91% for
individual motility, 2.83 and 2.95(x109) sperm/ml for sperm cell
concentration, 76.90% and 84.73% for live spermatozoa. The breed had
a significant effect on live cell percentage and mid-piece abnormalities.
The summer exerted a significant effect on all semen characteristics and
sperm cell abnormalities except semen volume. Reaction time was
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16.14 for Hamari and 20.10 seconds for Kabashi rams. Breed and
winter season exerted significant effect on RT. Mean values of SC were
33.12 and 31.38 cm for Hamari and Kabashi rams, respectively. Breed
exerted a significant effect on SC but the season of the year did not.
The ram semen was diluted with extenders containing glucose,
egg yolk and glycerol and buffered with Tris-citrate. The post-thaw
progressive motility were 56.75% and 56.19% and the incidence of total
rejected batches of semen before and after freezing were 46.15% and
38.79% for Hamari and Kabashi rams, respectively. Breed and season
had no significant effect on post-thaw motility of frozen semen, but
there was a significant difference (P< 0.05) between the two breeds and
summer season in the rate of rejected semen after freezing (15.38% vs.
6.35%); better results were obtained with Kabashi breed. One hundred
and fourteen ewes, aged 2-3 years, were given vaginal progestagen
impregenated sponges for 14 days and were injected with 500 i.u PMSG
on day of sponge removal for oestrous synchronization. All females
were inseminated in the cervix with 0.5 ml frozen-thawed semen, 55
hours after sponge removal. There was no significant difference in
pregnancy rate after AI and in the lambing rates and prolificacy between
Hamari and Kabashi breeds.
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The present study showed that the two breeds are continuous
breeding animals and they are capable of producing semen of good
quality all the year round. The semen collected was suitable for freezing
and the results obtained after cervical insemination will positively
contribute to intensive sheep production.
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المستخلص
على النشاط الجنسي وخواص السائل مواسمال هدفت التجربة لدراسة تأثير الساللة و
شملت الدراسة اختبار قابلية تجميد السائل . المنوي لساللتين من الضأن الصحراوي السوداني
سنوات 4- 2آان عمر الخراف. المنوي ومن ثم خصوبة النطف المجمدة لتلك الساللتين
آل الخراف . آجم للحمري والكباشي على التوالي61.2 و 73.6وزنها عند بداية الدراسة و
خراف حمرية 6قذفة منوية من 293 ت جمع. تمت تغذيتها ورعايتها تحت ظروف مماثلة
مل لحجم 1.23و 1.31 آان متوسط خواص القذفات المنويةت النتائج أنوضحأ .آباشية 3و
2.83، للحرآة الفردية % 68.11 و %67.91، درجة للحرآة الجماعية 2.73و 2.37 ،القذفة
. للحيامن الحية%84.73 و 76.90%، لعدد الحيوانات المنوية في المليلتر)109x 2.95 (و
آما أثر . أثرت الساللة تأثيرا معنويا على نسبة الحيامن الحية وعيوب عنق الحيوانات المنوية
على آل تشوهات الحيوانات المنوية وخصائص السائل المنوى فصل الصيف تأثيرا معنويا
ثانية 20.10 و 16.14 للحمرىوجد أن متوسط التفاعل الجنسي . ماعدا حجم القذفة
33.12متوسط محيط المناسل بلغ . تأثيرا معنويا على زمن التفاعل الجنسى لهالشتاء.لكباشيل
ة لها تأثيرا معنويا على هذه الصفة أما فصول وآان السالل. للكباشىسم 31.38وللحمرى
.السنة فلم يكن لها تأثير معنوى
صفار البيض والجلسرول، تم تخفيف السائل المنوى فى مخففات تتكون من الجلكوز
اإلذابة و التجميدمتوسط حرآة الحيوانات المنوية بعدبلغ . السترات-ومعادلتها بالترس
للحمري %38.79و %46.15 ات المبعدة قبل وبعد التجميد العيننسبةو%. 56.19 و56.75%
لم يكن للساللة وفصول السنة تأثير معنوى على حرآة الحيوانات .والكباشي على التوالي
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البيانات توضح وجود فروق معنوية بين الساللتين وفصل الصيف المنوية بعد اإلذابة ولكن
ل على نتائج جيده مع الحصو )%6.35 مقابل 15.38(في نسبة العينات المبعدة بعد التجميد
اسفنجات مهبلية مشبعة بهرمون سنوات 2 - 3نعجة في عمر 114 أعطيت.لساللة الكباشي
وحدة عالمية من مصل الفرس الحامل 500يوما ثم حقنت 14 البروجستيرون لمدة
PMSG) ( 0.5 عنق الرحم ب فىلقحت آل النعاج . عند ازالة االسفنجة وذلك إلحداث الشبق
ا فرقم يكن هناكل. ساعة من ازالة االسفنجة المهبلية55 مذاب بعد -مل سائل منوى مجمد
.ين الحمري والكباشيب والخصوبة نسبة الوالداتو في نسبة الحمل بعد التلقيح امعنوي
و وانتاج سائل منوي ذمستمرالتناسل القدرة على الامتين لهل أن السال الدراسة أوضحت
والنتائج المتحصل عليها من التلقيح في .وامكانية تجميده جيده على مدار العام مواصفات
. لتوليد األغنامواعدة تشجع فى تطبيقها على نطاق واسعأعطت نتائج عنق الرحم
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Introduction:
The sheep population in the Sudan was estimated to be 51.067
million heads, with an annual increasing rate of one percent (Ministry of
Animal Resources: 2008).
Mcleory (1961) classified the sheep of Sudan into eight distinct
ecotypes according to locality, tribe and origin. Of these ecotypes, the
Sudan Desert sheep constitutes 65 percent of the sheep population in the
country. These animals are normally found in the desert and semi-desert
areas of the Sudan (annual rain-fall ranging between 75 and 300
mm).The desert sheep dominate the export market of live sheep and
mutton. However, Hamari and Kabashi sub-types are dominant over the
other Desert sub-types and collectively the Desert sheep play an
important role in the national economy either through satisfying the
local need or through their export revenue of hard currency (cited
Babiker et al.,1988).
Most of sheep population, in the Sudan, is raised under the
traditional nomadic system of management. Thus they are exposed to a
wide range of adverse climatic and nutritional stresses in addition to
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endemic diseases. Official and private sectors exert efforts to shift
towards intensive modern system were limited and short-lived.
In any breeding programmes, the use of superior sires could have
a direct effect on the reproductive and productive traits of the resultant
progeny, and this could satisfy both the producer and consumer interest.
Breeding soundness examination is the first step in management of rams
for optimum breeding performance. Selection of the most fertile ram
may not only improve the lamb crop but also allows for a large ewe to
ram ratio and thus reduces the cost of keeping many rams.
One of the keys to successful artificial insemination (AI)
programmes is to know when to inseminate the female. There are
various methods of oestrous synchronization which have been used
successfully in sheep. Utilization of progestagins and follicular
stimulants (eCG) for controlling the oestrous cycle and for inducing
out-of-season breeding has been commercially available for many years.
When oestrus synchronization is employed, extra rams are required to
mate the ewes over a very short period, AI will reduce the number of
rams needed and facilitate a faster insemination.
The progress made in cryopreservation of ram semen has opened
the possibility for conservation and utilizing of frozen semen of elite
rams in sheep improvement programme. Though, AI with frozen semen
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in sheep has not been well spread in the world, probably because of the
intensive nature of sheep breeding and relatively poor fertility results
obtained when frozen semen is used for cervical insemination
(Söderquist, 1999).
However, the knowledge gained in studying the reproductive
pattern of rams is useful when deciding the best period to breed ewes
with high quality semen or the best periods to harvest ram semen meant
for freezing for AI purposes.
Previous studies on the measurement of the reproductive
capacity, seasonality and fertility of Sudan Desert sheep have been
carried by many investigators (Galil and Galil, 1982; Alsayed, 1996;
Suhair, 1997; Manahil, 1999).
Introduction of modern techniques such as oestrous
synchronization and AI with fresh diluted semen in the Sudan sheep is
recent and confined to research institutes (Alsayed, 1996; Manahil,
1999; El Mubark, 2001; Alsayed, 2001). However, wide-scale adoption
of AI using fresh or frozen semen and monitoring of the success rate of
this procedure remained to be investigated.
The basis of production of ram semen in the laboratory is to train
the ram to ejaculate in an artificial vagina. Then the semen is collected,
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grossly and microscopically evaluated. Then the semen is process,
frozen and stored until insemination.
The objective of the study:
(1) To study the influence of the breed and season on semen quality
and sexual performance.
(2) To assess the relationship between semen volume, sperm cell
concentration, body weight and scrotal circumference
measurement of the two breeds.
(3) To compare the freezability of the semen of the two breeds in
different seasons of the year.
(4) To study the conception rate in the oestrous synchronized ewes
inseminated with the frozen semen obtained from the two breeds.
It is hoped that the production of frozen semen and the
understanding of the seasonal impacts on the reproductive performance
of the Hamari and Kabashi breeds would assist to formulate a rational
breeding programme for genetic improvement at the appropriate time of
the year and at different localities using semen from the same proven
sire. This will offer significant opportunities to reach maximum
exploitation of this superior genetic material through this technique
which will be introduced for the first time in the Sudan.
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1
Literature Review
2
1. Reproduction in sheep:
1.1 Libido and mating behaviour in rams:
The term libido is commonly used to describe sex-drive in entire
male animals (Fraser, 1974). Libido is primarily dependent upon
androgenic steroid hormones, which allow mating, and aggressive
behaviour to occur, as well as maintaining the function of all parts of the
male reproductive system (Arthur et al., 1998). The most obvious
expression of libido is during the copulatory behaviour which can only be
expressed post-puberally. However, the actions of copulation may be
mimicked by pre-puberal males and by females while under oestrogenic
influence (Chenoweth, 1981). Ram exhibit libido by behavioural
components such as nosing the female perineum, nudging, flicking of
tongue, striking out with a fore-limb and low pitched bleats (Nabil et al.,
2006). Although these activities may all is described as sexual behaviour
the following definition can be used (Chenoweth, 1981):
Libido: the willingness and eagerness of a male animal to mount and
attempt service of a female.
Mating behaviour: The behaviour of the male animal in the periods
immediately before, during and after service.
Sexual performance tests have been used as a tool to predict ram
performance (Kilgour, 1993; Stellflug et al., 2006). Libido and mating
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ability are important traits which can affect production significantly
(Chenoweth, 1981). In intensive breeding programmes a single ram can
breed 8–10 ewes daily, or well over 100 ewes in the course of a single
oestrous cycle (Chenoweth, 1981). In some trials measuring the
reproductive capacity of rams, great variations have been reported
between individuals (Hulet et al., 1962). However, rams tend to distribute
their services among receptive ewes through the choice of ewes with
which they have not mated before or with which they have mated less
frequently (Chenoweth, 1981). In closely, confined groups of ewes,
dominant rams inseminate the majority of ewes and may prevent other
rams from breeding (Hulet et al., 1975).
Different methods have been employed to assess sex-drive in rams
including reaction time to service, the number of services within a limited
time period and libido score (Chenoweth, 1981). Changing the stimulus
female for second ejaculation had no effect on the semen characteristics
or the inter-ejaculation interval. However, this increased the number of
mounts needed to achieve a second ejaculation, while preventing physical
contact with the stimulus animal after the first ejaculation reduced the
number of mounts (Lezama et al., 2003).
Yearling rams engaged in more leg-kicking bouts and showed
higher mounting frequency than two years Awassi rams (Kridli and Said,
4
1999). Sexual performance of rams that differ in age (maturation) and
sexual experience, when they have one or two relatively brief exposures
to oestrous ewes can bring the sexual performance of virgin rams up to
the levels comparable to that of experienced males (Price et al., 1991).
However, exposing sexually naive rams to oestrous ewes before the
breeding season may be necessary to improve their sexual performance
(Kridli and Said, 1999). Trejo et al. (1990) reported that, breed of ram
had no significant effect on the interval from contact with a female to
ejaculation (reaction time), the interval between the first and second
ejaculation (recuperation time), the total number of services per ram in 30
minutes or the number of mounts per ejaculation. Lezama et al. (2001)
found that, spreading semen from a different ram in the vulva of the
stimulus animal was more efficient than spreading semen of the same
male or the absence of semen, in shortening the inter-ejaculatory interval,
but no relationship was found between this effect and semen
characteristics.
It is well known in temperate zones that ram sexual activity is
subjected to seasonal variations and the intensities varies from breed to
breed. These variations can affect all the components of reproductive
function particularly semen quality and its fertilizing ability (Salamon
and Robinson, 1962; Smyth and Gordon, 1977; Dufour et al., 1984;
5
Melpomeni et al., 2004). However, rams have been reported to show
more sexual activity in autumn, while in another study it was observed
that some decline occurred in the libido of Targhee rams during the
spring (Chenoweth, 1981). However, Mickelsen et al. (1981) suggested
that, if the sex-drive were normal in some breeds of rams, perhaps twice a
year breeding would be realistic, if fertility of ewes were normal. Ibrahim
(1997) found that, the mean reaction time for local and cross-bred rams
(Local x Chios) raised in United Arab Emirates were reported to have an
average RT of 43.7 seconds which affected mainly by season.
Furthermore, he showed that, the reaction time was shorter (20 seconds)
in summer and longer (72 seconds) in autumn, indicating that the high
temperature in summer did not decrease sexual activity of the rams.
It has been reported that rams fed on a high protein supplement
displayed a more intense sex-drive than rams fed on a low protein
supplement (Chenoweth, 1981).
1.2 Semen:
Semen is the normal discharge of the reproductive organs
ejaculated by the male. It contains sperm cells and seminal plasma. The
seminal plasma is a mixture of fluids secreted mainly by the vesicular
glands, with lesser contribution from the testes, epididymides, deferent
ducts and the accessory glands (Herman et al., 1994). The seminal plasma
6
of rams is clear or opaque fluid, usually isotonic, neutral fluid with a pH
close to 7.0 (Evans and Maxwell, 1987). However, prolonged exposure of
spermatozoa to seminal plasma has been found to have an adverse effect
on sperm function. The loss of sperm heterogeneity and membrane
integrity during treatment and that the freezing-thawing process, were
less apparent when seminal plasma had been removed from semen
samples before freezing (Ollero et al., 1997). Therefore, the separation of
mammalian spermatozoa from seminal plasma is a practice routinely used
in the laboratory and in assisted reproductive technology application
(Perez et al., 2001). In contrast, the seminal plasma components
prevented and reverted cold-shock damage on sperm membrane and
improved the viability and fertility of frozen-thawed sperm (Jobim et al.,
2005). Moreover, the seminal plasma proteins that bound to the sperm
surface may be responsible for sperm membrane stabilization
(Dominguez et al., 2008).
1.3 Semen characteristics:
The routine semen evaluation for artificial insemination application
relies on assessing a number of parameters such as sperm motility,
concentration and morphology for prediction of fertility in the male
(Correa and Zavos, 1994). The hypo-osmotic swelling test (HOS-test) is
used to evaluate the sperm functional status (integrity) and permeability
7
(Moussa, 1999 and Zeidan et al., 2006).The development of computer-
assisted sperm analysis (CASA) allows an objective assessment of
different cell characteristics: motion, velocity and morphology (Sancho et
al., 1998 and Verstegen et al., 2002).
1.3.1 Colour and consistency of semen:
The normal colour of semen is milky–white or pale cream (Evans
and Maxwell, 1987). Some rams can have a very yellowish colour of a
normal creamy ejaculate; this is normal for those rams which have high
levels of riboflavin in the ejaculate and which is a harmless pigment
(Nabil et al., 2006). Mature ram during breeding season would have a
thick, creamy consistency semen samples. A sub-normal ram may
produce a sample looks like cloudy water or thin milk (David, 1994). Any
abnormal colour, odour or foreign material such as hair, faecal particles,
bedding and dirt indicate poor hygienic quality (Nabil et al., 2006). The
presence of blood or pus flakes may indicate affection in reproductive
tract (Nabil et al., 2006). Dabas et al. (1997) reported that, most of the
semen samples collected from six Potanwadi rams, aged 2-4 years, during
the pre-breeding, breeding and post-breeding seasons, showed creamy
thick (48%) or creamy white and thick (42%) consistency. The normal
semen colour of Sudan Desert sheep was found to be creamy-white
varying from slightly thick to very thick (Galil and Galil, 1982).
8
1.3.2 Volume of ejaculate:
Normal volume of ejaculate in adult rams (1.5 years of age or
older) is of 0.8 to 1.2 millilitre (ml), with a mean of one ml (Foote, 1974;
Gomes, 1977). However, Herman et al.(1994) reported that, the average
volume of ram ejaculate was about 1.0 to 1.5 ml. Dabas et al. (1997)
found that the ejaculate of Patanwadi rams averaged 0.96+0.03 ml. The
Sudan Desert sheep showed an average of 0.99 ml with a range of 0.82 to
1.22 ml for the ejaculate volume (Galil and Galil, 1982). Moreover, the
average ejaculate volume that was reported by Alsayed (1996) in the
same breed was 1.37 ml, while that found by Manahil (1999) averaged
1.62 ml. Chiboka (1980) found that, in the African dwarf rams , there was
a significant week seasonal effect on the volume of semen during the dry
period but not in the rainy season.
1.3.3 Concentration of spermatozoa:
Good quality ram semen contains 3.5 to 6.0 billion spermatozoa
per ml (Evans and Maxwell, 1987). However, Herman et al. (1994)
reported that, the average ram ejaculate contains from 1 to 3 billion
sperm. Cameron et al.(1987) found that, the mean number of
spermatozoa ejaculated by an individual ram varied from 140 million to
1050 million, following depletion of the epididymal reserves of
spermatozoa. Galil and Galil (1982) found that, the sperm count of the
9
Sudan Desert sheep was 2.452 billion per ml with a range of 1.85 to 3.4
billion. However, Alsayed (1996) reported that the average concentration
of spermatozoa was 2.5 billion per ml while that reported by Manahil
(1999) was 3.4 billion per ml for the same breed.
1.3.4 Sperm motility:
The normal percentage of motile spermatozoa is around 75 percent
with a range between 60 and 80 (Gomes, 1977). In the Sudan Desert
sheep, the sperm mass motility score was found to be 4.12 with a range of
3.19 to 4.8 scores and individual motility was 79.5% with a range of 63%
to 89% (Galil and Galil, 1982). Manahil (1999) reported that, the mass
motility score of Sudan Desert sheep ranged between 4 and 5 (average
4.3 + 0.5). The individual motility of Sudan Desert sheep ranged between
75–90% (average 83.9+5.3%).
1.3.5 Sperm cell morphology:
The evaluation of the incidence of morphological abnormal
spermatozoa in an ejaculate is important. Only sperm cells with intact
acrosome and normal structure are fertilizes the ovum. The normal
mature ram spermatozoa head is approximately 8 micrometer (µ m) long,
4 µ m wide and 1µ m thick. The mid-piece is approximately 14 µ m long
and the tail is 40 to 45 µ m long. The entire sperm measures
approximately 65 µ m (Sorensen, 1979).
10
Under electronic microscope, the sperm head has a regular oval
shape with a pronounced apical ridge, a nuclear ring and two
protuberances on the lateral rim. The acrosome consisted of dark and
light zones, and the tail tapered off posterior to Jensen’s ring (Massanyi,
1989).
Many studies have shown that the initial ejaculate contains a
greater number of abnormal cells; these abnormalities consist, for the
most part, of head malformations and proximal cytoplasmic droplets,
which indicate incomplete spermatogenic activity and incomplete
epididymal function (Skinner and Rowson, 1968). The percentage of
abnormal spermatozoa showed larger variations between semen
collections and between different sires (Colas et al., 1990). The presence
of immature forms of sperms may indicate that a ram is being
overworked (David, 1994). Dabas et al. (1997) found that, the average
abnormal spermatozoa of Patanwadi rams were 7.9+0.01 percent.
However, Galil and Galil (1982) observed that, the abnormal
spermatozoa of the Sudan Desert sheep was 3.5 percent with range of
2.40 to 4.99.
11
1.4 Factors affecting semen production:
The main factors which may affect the quality and quantity of
semen include; age of the ram, nutrition, testes size, environment,
frequency of semen collection and breed.
1.4.1 Age of the ram:
In sheep as in most other species, the quality and quantity of semen
is strongly influenced by age of the animal (Demrici, 1993; Rege et al.,
2000). The age at the first appearance of spermatozoa in ejaculates in
three breeds of rams (Chios, Serres and Karaguniki) averaged 20.1+0.31,
20.4+0.37 and 20.6+54 weeks and body weight averaged 36.4+0.56,
36.5+0.7 and 34.9±0.99 kg respectively (Alexopoulos et al.,1991).
However, the semen volume, sperm motility score and sperm
concentration increased with increasing age, while the percentage of dead
and morphologically abnormal spermatozoa decreased (Alexopoulos et
al., 1991).
1.4.2 Nutrition:
The influence of nutrition on reproduction is mediated via the
effects of dietary constituent on the hypothalamic–pituitary gonadal axis
in both ewes and rams (Brown, 1994). The extensive supplementation of
the ram-lamb during intra-uterine and post-natal life could result in higher
testicular growth and sperm production when they become adults (Bielli
12
et al., 2000). However, over-feeding of young rams could be detrimental
to their fertility (Fourie et al., 2004). The morphology of the seasonal
testis regression of Corridale rams is not easily reverted by the improved
feeding, so the enhancement in testis development when pre and post-
pubertal rams grazed improved pasture was short-lived. The tendency to
increase Sertoli cells numbers elicited by treating animals with improved
pasture plus grain supplementation from foetal to pre-pubertal life might
result in larger-lived consequences to spermatogenic capacity (Bielli,
1999).
Changes in the nutrition of mature rams lead to profound responses
in testicular size and therefore the rate of production of spermatozoa.
These effects are largely due to changes in the size of the testes and
seminiferous tubules and in the efficiency of spermatogenesis (Fernandez
et al., 1993; Martin and Walkden-Brown, 1995).
In mature rams, however, there is a delay of six to seven weeks in
the response of spermatozoa numbers to diet, reflecting the time it takes
for the development of spherical spermatoid in the germinal epithelium to
fully mature spermatozoa in the distal caudal epididymides (Robinson,
1996). The effect of nutrition has been demonstrated by results which
have shown that daily sperm production tends to decline as the level of
energy and protein in the diet declines. The rate of sperm production per
13
gram of testis was approximately 40% greater in Merino rams maintained
on a high, rather than low plane of nutrition (Cameron et al., 1988).
Similarly, Oldham et al. (1978) achieved rates of spermatozoa production
of 13.6 billion versus 7.5 billion per day, through providing different
levels of nutrition. Martin and Walkden-Brown (1995) and Martin et al.
(1999) concluded that, nutritional effect can be divided into short-term
(10–20 days) effect that mainly act on the neuroendocrine system
(Stimulates Gn RH/LH pulse frequency) controlling testicular activity
and long-term (several months) effect that act on testicular growth and
sperm production. The increasing level of feeding by 30% of total
nutrients above the recommended level or 38% of the total protein,
increased semen volume per ejaculate and improved semen quality
(Khachirov, 1984). The addition of lysine at a rate of 4.36% and
methionine at a rate of 3.9 to 4.0% of the weight of crude protein, to the
diet of ram during the breeding season increased ejaculate volume by
17.8%, sperm concentration by 28.5%, sperm resistance by 17.1% and
fertilizing ability by 4.0% (Zpydnev and Orekhov, 1989).
Supplementation of the diet with 18–36 gm sun flower oil daily,
increased semen quality and quantity, motility increased by 11.3–12.4%
and conception rate using frozen semen was 5.6–10.7% higher for
supplemented rams (Davidenko et al., 1990). Rams given diet containing
12% cotton-seed meal did not differ from that of the control group in
14
their body weight, semen volume, semen motility and concentration of
spermatozoa, but the percentage of abnormal spermatozoa was higher in
the cotton-seed fed rams (17%) than the controls. The histological
examination of the testes showed that rams fed cotton-seed meal had
larger lumen diameter, fewer number of cell layers and lower wall
thickness in seminiferous tubules, and smaller size of Sertoli and Leydig
cells than the control rams (Arshami and Ruttle, 1989). In rams fed diets
containing gossypol, ejaculated sperms appear normal under light
microscopy, but the integrity of the membrane of sperm cells may be
damaged due to the extensive damage of germinal epithelium (Randel et
al., 1992).
The effect of vitamin E and selenium added to the ration was
studied by Gokcen et al. (1990); they found that, the semen
characteristics and acrosomal integrity were significantly better in
supplemented animals than the controls. Tenlibaeva (1991) observed that,
the addition of vitamins A and E to the diet has a positive effect on sperm
production and on the fertility level of ewes.
1.4.3 Testes size:
Testes are the primary organs of reproduction in the male. They are
two in number, and are carried outside the body wall in the scrotum
(Herman et al., 1994). There are no significant differences between the
15
weight and volume of the left and right testes and epididymides (Colyer,
1971; Martinez et al., 1994). The testes have at least two functions:
(1) Production of spermatozoa (male germ cells).
(2) Production of endocrine substances or male hormones
(particularly testosterone) that markedly affect the
development and behaviour of the male (Herman et al.,
1994).
Testis size has been regarded as a trait of choice in male (Matos and
Thomas, 1992). Scrotal circumference has been suggested as an indicator
of seminal performance and male libido as well as the age at puberty. The
testicular development in young rams was found more closely associated
with body weight than with its age (Matos and Thomas, 1992). Master
(1988) suggested that, the young ram to be used for breeding should have
scrotal circumference more than 30 centimetres (cm) and that value of
35 cm is the normal for two years old rams. However, the scrotal volume
decreased by 18–26% in rams mated for 4–6 weeks, with only part of the
decrease being accounted for by the 4–12% decrease in live weight
(Knight et al., 1987).
In many studies testicular measurements have been evaluated and
related to some seminal parameters, usually sperm concentration and
sperm motility (Ruttle et al., 1982; Gipson et al., 1985). Testicular
16
circumference is directly related to sperm production and rams with
larger testes produce more sperm (Langford et al., 1998). However, the
ejaculate volume was found to be significantly correlated with testis size
(Vijil et al., 1987; Demirci, 1993). Rege et al. (2000) found that , genetic
correlations of scrotal circumference with semen volume (0.55±0.11),
mass motility (0.62±0.20), individual motility (0.54±0.12), concentration
(0.25±0.04) and proportion of abnormal spermatozoa (-0.75±0.24) in 12-
month for Menz and Horro old rams. The selection based on this trait,
have appreciable favourable correlation response in semen quality and
spermatozoa production. Follicular stimulating hormone (FSH) levels, at
all ages, were correlated negatively with scrotal size; these findings
indicate that FSH levels in ram lambs may be useful for predicting adult
testis function (Langford et al., 1998). Colas et al. (1990) concluded that,
lambs could inherit seasonal variation in testicular diameter.
In 3.5 to 4 years, Pelibuey rams, the daily sperm cell production
was found to be 27.5+1.5 million sperm cells per gram of testicular
parenchyma and 5094+366 million sperm cells per testis. Epididymal
sperm reserve accounted for 28.1+1.3 billion spermatozoa per organ
(Martinez et al., 1994). However, Cardoso and Queiroz (1988) reported
that, in Brazilian hairy rams, the mean values obtained for sperm
production were 22.8+8.9 million sperm cells per gram of testis
17
parenchyma and 2.2+1.3 billion sperm-cells per testis per day. The
efficiency of spermatogenesis and degeneration of different
spermatogenic cells under normal conditions of the environment in rams
was 47.58 percent, since one spermatogonium produces 30-45 spermatids
against the expected number of 64 (Bilaspuri and Guraya, 1986). The
spermatogenic cycle was estimated to take 42.28 days from the first
spermotogonial mitosis of A1 spermatogonia to the liberation of
spermatozoa (Cardoso and Queiroz, 1988).
However, clinical examination of genital organs seems to offer a
reliable source of information about rams, and may be considered
adequate in sheep breeding (Weirzbowski and Kareta, 1993). The
consistency of testis is described as firm and elastic. Although there are
grades within these dimensions, (firm and elastic) deviations from this
including both soft and flabby and firm and dull, are also used. Any
enlargements of the epididymis should receive due attention (Gallway,
1966).
1.4.4 Environment:
Photoperiod and temperature are two of the most important
environmental factors influencing reproduction of sheep. Seasonal
variation in sexual activity among small ruminants depend on latitude and
day length. Melatonin secreted during the night by the pineal gland
18
enables the animal to determine the length of the day. This hormone can
be used to advance mating season of sheep and increase its fecundity. The
use of artificial photoperiodic variations and melatonin eliminates or
alters seasonality of sperm production (Chemineau, 1993).
1.4.4.1 Temperature:
The effect of high temperature on the reproductive performance of
ram is well established. The exposure of the animal to hot temperature
results in a change in the morphological profile of the semen (Zamba,
1971; Marai et al., 2007). Under heat stress, libido, semen production and
fertilizing ability of males are significantly reduced. However, in females,
heat stress reduces the oestrous period and level of fecundity and
increases early embryonic mortality (Chemineau, 1993). In sub-tropical
conditions, the environmental temperature significantly affect sperm
motility, sperm concentration and sperm number per ejaculate (Daader et
al., 1987). Howarth (1969) suggested that the deleterious effect of
elevated temperature on sperm quality is due to damage sustained during
the late stages of spermatogenesis and would further attest to the apparent
resistance of epididymal spermatozoa to the effect of heat. The resistance
of epididymal spermatozoa to the effects of heat could be due to their
lower metabolic rate compared to dividing cells. Ibrahim (1997) observed
that, the best semen quality of local rams in United Arab Emirates was
19
obtained in winter, but the semen collected in other seasons is also judged
to be of satisfactory quality. Galil and Galil (1982) concluded that, Sudan
Desert sheep appeared to be able to breed through-out the year and their
spermatogenesis was not seriously affected by high environmental
temperature.
1.4.4.2 Photoperiod:
The hypothesis proposes that melatonin secreted by the pineal
gland acts at separate sites in the brain and pituitary gland to regulate
gonodotrophin and prolactin secretion, and these responses mediate the
effects of photoperiod on the time and characteristics of the seasonal
reproductive cycle. The pituitary gland is the proposed site of action of
melatonin for the control of prolactin secretion (Lincoln, 1999). The
seasonal cycle of reproduction is a result of a complex interaction of at
least four distinct mechanisms (Pelletier, 1996):
• Light, which stimulates gonadotrophic activity mainly under
conditions of natural or artificial decreasing of day length?
• The intensity of gonadal steroid negative feed-back, which
varies depending on the day length.
• The existence of an inherent annual pattern of reproduction.
• The existence of photosensitive phase during which light
stimulates gonadotrophic activity.
20
Thus gonadotrophins and gonadal steroid hormones act in concert with
prolactin to determine the functional state of the reproductive axis. This
interaction potentially affects the functional state of the gonads, accessory
sex glands, secondary sex characteristics and the expression of sexual and
aggressive behaviour during sexual cycle in seasonally breeding
mammals (Lincoln, 1999).
Further studies, showed that, light regime with alternating 1-2
month periods of increasing and decreasing day length stimulates the
gonadotrophins in alternate months and maintains permanent sexual
activity (Malpaux et al., 1995). With this regime, plasma testosterone
never reaches the levels which are considered inhibitory at the end of the
breeding season (Pelletier, 1996). This alternating light regime has
maintained the quantity and quality of semen at levels normally observed
during the breeding season (Pelletier, 1996). Although, the treatment with
melatonin may be considered as an alternative to photo-stimulation for
the preparation of rams for out-of-season breeding (Hanif and Williams,
1991).
1.4.5 Frequency of semen collection:
Frequent ejaculation (4, 5 or 8 times daily) for 14-36 days duration
produced a decrease in semen volume by 25–53%, sperm motility by 19-
36% and sperm concentration by 19–55% (Thwaites, 1995). Semen of the
21
second ejaculate was significantly less in volume, greater in motility,
closer to neutrality and less per cell concentration when two separate
successive ejaculates were obtained, however, characteristic of the
second ejaculate fell within the normal range of high quality semen
(Ibrahim, 1997).
1.4.6 Breed:
Breed differences in semen quality and quantity was reported in
many studies. Langford et al. (1998) found that, Canadian rams produced
more sperm per ejaculate than Finnish Landrace (7.0 billion compared to
3.3 billion). Fernandez et al. (1993) observed that, the Corriedale rams
had lower semen production and sperm concentration and higher
incidence of sperm abnormalities than rams of other breeds (Polwarth,
Merilin and Merino). However, Vivanco (1989) studied the influence of
breed and reproductive characters of rams under high altitude conditions.
He found that, Criollos tended to have better semen quality than
Corriedales and Junins at first ejaculation and at all ages. Abdel-Rahman
et al .(2000) reported that the Somalian ( Barabari ) and Sudan (Sawakni)
breeds showed reduction in sperm concentration, percentage of live
spermatozoa, highest percentage of individual motility and the highest
concentration of sodium (Na) chloride (Cl) and inorganic phosphorus (P)
in the whole semen, seminal plasma and spermatozoa, while superior
22
sperm density, percentage live spermatozoa and seminal concentration of
potassium (K) and calcium (Ca) were evidenced in the Najdi, Naemi and
to lesser extent, in Merino breeds. However, no breed difference was
observed in semen quality between local Emirate breed and its cross with
Chios breed (Ibrahim, 1997).
1.5 Seasonal effect on reproduction:
Reproduction in sheep is a complex mix of environmental factors
and hormonal responses in ewe and ram. Several studies have indicated
that semen production is influenced by seasonal changes (Colas and
Brice, 1976; Jennings, 1976; Loubser and Van Niekerk, 1983; Mandiki et
al., 1998). Dufour et al. (1984) showed that, the seasonal variation can
affect all the components of the reproductive traits, being lowest during
the summer and highest during the fall. Significant differences between
the breeding and non-breeding seasons were obtained from the blood.
The activities of the acid phosphatase, aspartate and alanine, amino-
transferase and concentration of total protein and sialic acid, all were due
to a significant decrease in values during breeding season (Karcheva and
Baulov, 1989). Sheep from temperate latitudes display seasonal variation
in their reproductive performance which is controlled by the annual
fluctuation in the day length (Malpaux et al., 1995). There is a seasonal
variation in the dimension of the reproductive organs of the animals and
23
that during the breeding seasons, testicular size and weight are intimately
correlated (Nwoha, 1996). The seasonal variation in testis size has been
recorded, but rams appeared to be sexually active when introduced to
oestrous ewes out of season (Dyrmundsson et al., 1989; Melpomeni et
al.2004). The magnitude of seasonal variation in semen quality was not
sufficient to prevent rams from being used for breeding throughout the
year (Gündogan, 2006). Berg (1999) reported that, in rams there were no
total seasonal limitation of the sexual function, but the gonadal activity is
somewhat reduced in the period from early spring to next autumn. Dabas
et al. (1997) reported that, season had a significant effect on mass activity
of semen, pH, sperm concentration and percentage of dead and abnormal
spermatozoa of Patanwadi rams. In sub-tropical conditions,
environmental temperature significantly affects sperm motility, sperm
concentration and sperm number per ejaculate (Daader et al., 1987). In
West African dwarf rams, semen volume, sperm cell concentration did
not differ between dry and wet seasons, whereas, percentage of
progressive motility, total abnormalities and live sperm in dry season
were different from those in the rainy season which showed better quality
in these criteria (Chiboka, 1980). Seasonal variation in testicular
morphology between Corriedale rams subjected to different feeding
levels was observed. These findings suggested that nutritional factor
contributed, at least partly, to the differences in variation observed (Bielli
24
et al., 1997). The season exerts a significant influence on semen
freezability in Leccese ram, with the best performance occurring in
summer and autumn period, corresponding to the breeding season in
temperate zones (D´Alessandro, 2003).
1.6 Semen collection:
Semen may be obtained by one of three methods:
(1) Collection from the vagina after mating.
(2) Artificial vagina.
(3) Electrical stimulation (Electro-ejaculation).
1.6.1 Collection from the vagina after mating:
The collection of semen from the vagina of a recently bred ewe is
possible by use of a syringe with soft nozzle. However, such semen is
small in quantity, mixed with mucus, sloughed cells, debris and micro-
organisms. This procedure is no longer in practice (Herman et al., 1994).
1.6.2 Artificial vagina (AV):
The AV is the best method for collecting natural ejaculate. Its also
allows evaluation of sperm quality, potency (sperm delivery to the
vagina) and libido of the animal. Moreover, when the AV is used for
semen collection, rutting behaviour of rams appeared to be normal
(Sackman and Schone, 1990). The AV provides suitable temperature,
pressure and lubrication (Evans and Maxwell, 1987). The AV consists of
25
a medium–hard rubber casing about 20 centimetres (cm) in length and
about 4 cm in diameter, with a soft rubber lining, about 25 cm in length
placed in the casing and each end folded over the edges and held in place
by rubber bands (Herman et al., 1994).
The temperature of the AV at the time of collection should be
between 41° and 44°C. If the temperature drops below 40°C or goes
above 45°C, it may inhibit ejaculation. The required pressure in the AV
may vary from one ram to another, but with a little experience, an
operator will soon determine the proper pressure (Herman et al., 1994).
As the ram mounted the teaser, the penile sheath is grasped by the
operator and the penis is directed into the AV. The collector must be alert
because the ram moves very quickly and makes a rapid single thrusting
motion (Sorensen, 1979).
1.6.3 Electro-ejaculation:
Electrical stimulation works successfully in sheep and it is similar
to the procedure described for the bull. Small rectal probe for rams are
commercially available for this purpose. Semen obtained by electrical
stimulation is usually few in sperm number and thinner in consistency
than that obtained with an AV (Herman et al., 1994). In addition,
electrical ejaculation resulted in a larger volume of semen, higher pH,
lower concentration and gross motility (Matter and Voglmayer, 1962).
26
However, there was no difference in fertility found between ewes bred
with semen collected by AV or electro-ejaculation (Hackett and
Wolynetz, 1982).
The rectal probe is pressed forward and downwards against the
floor of the pelvis by an assistant and short stimuli (3-8 seconds) are
applied at 15-20 seconds intervals. After 3-5 bouts of stimulation,
accessory glands secretion will flow, followed by semen (Evans and
Maxwell, 1987). If the penis is not extended, it may be necessary to push
the sheath and prepuce back a long the shaft to expose the penis and the
glans before semen collection (Sorensen, 1979). Ejaculation (usually
without erection) generally takes place in the rest phase between
stimulations and it may be necessary to milk the sheath to retrieve the
sample (David, 1994).
However, about 20 semen collections a week can be taken from a
ram during the breeding season, but only four or five a week out of
season (David, 1994).
1.7 Semen dilution:
Semen extenders provide some nutrients for the metabolic process
of sperms, protect against cold shock, buffer the effects of toxic and
acidic substance produced by sperm metabolism and aid in maintaining
the proper osmotic pressure and mineral balance (Herman et al., 1994).
27
Semen extenders usually contain a buffer such as sodium citrate
dehydrate or Tris (Tri- hydroxy methyl amino methane); egg yolk or
milk, which contains macromolecules that provide protection against cold
shock; simple sugars, such as glucose and fructose, that serve as a source
of energy for sperm; glycerol which is added as a cryoprotectant to
reduce the intercellular formation of ice crystals and solute effects during
the freeze-thaw process and antibiotics such as gentamycin, tylosin which
inhibit a variety of micro-organisms found in semen (Herman et al.,
1994). The addition of glycerol at 5˚C results in better sperm survival
than addition at 30˚C, although the difference is only small (Fisher and
Fairfull, 1989). It is due to ability of glycerol to pass through the
membrane at 30˚C. Cell membranes are less permeable to glycerol at 4˚C
so it has a less toxic effect (Critser et al., 1988). Lipid-binding proteins
present in seminal plasma (sp proteins) induce cholesterol and
phospholipids removal of sperm membrane which is detrimental to sperm
preservation. Low-density lipoproteins present in egg yolk interact with
lipid-bind proteins which minimize the cholesterol and phospholipids
removal. Skimmed milk, which is devoid of lipoprotein, also protects
sperms during storage by milk casein (Annick and Puttaswamy, 2006).
High egg-yolk concentrations reduce the post-thaw viability of ejaculated
spermatozoa in several species including rams (Watson and Martin,
28
1975). In addition, egg-yolk reduces the acrosome integrity of goat
spermatozoa (Abo agla and Terada, 2004).
Three extenders were used for processing of ram semen (Gil,
1999); each of them has two fractions for two-step extension
methodology, the first fraction without glycerol and the second with 14%
glycerol, thus resulting in a final concentration of 7% (v/v).
These extenders were prepared as follows:
(i) Powdered skim milk extender (A):
this extender consisted of reconstituted non fatty milk powder
(11% w/v) heated to 95°C for 10 minutes and then cooled to room
temperature. It is prepared into two fractions.
Fraction A-1 was prepared by adding egg yolk (5% v/v) to the
reconstituted milk and antibiotics (0.03 g penicillin and 0.04 g
streptomycin/100 ml.).
Fraction A-2 was prepared by adding egg yolk (5% v/v), glycerol
(14% v/v), 224 mM of fructose and antibiotics as described above.
29
(ii) Clarified powder skimmed milk extender (B):
It was prepared exactly like extender (A), but it was, somewhat,
clarified by centrifugation (3310g at 5°C for 20 min.). Centrifugation was
repeated with the supernatant.
Fraction B-2 was centrifuged before adding glycerol (14% v/v).
Only supernatant between the overlying lipoid and the compact pellet at
the bottom was used.
(iii) Tris–egg yolk-glycerol extender (C):
It consisted of 263 mM Tris, 85 mM citric acid, 73 mM fructose
and 20% (v/v) egg yolk.
Fraction C-1 consisted of extender C clarified by centrifugation (as
described for extender B).
Fraction C-2 consisted of C-1 plus glycerol (14% v/v). The
antibiotic mixture used in this extender is the same as that used in
extenders A and B.
Furthermore, Evans and Maxwell (1987) tabulated four different
formulae of Tris-citric acid-fructose-egg yolk-glycerol extenders for one
– step dilution methodology. One part of semen added to 1, 2, 3 or 4 parts
of extender according to the consistency of semen samples. Although,
the sperm viability was higher for semen processed in the clarified milk–
30
based extender compared to both milk-based extenders as well as the
Tris-citric acid fructose based extender (Söderquist, 1999). Significantly a
higher percentage of motile, progressive and membrane-intact
spermatozoa were recorded for milk than for the Tris extenders (Yániz et
al., 2008).
The current use of ingredients of animal origin, such as egg yolk,
milk and bovine serum albumin in semen extenders presents a risk of
microbial contamination, and has led to the search for alternatives. Such
an extender is commercially available for bull semen (Bioexcell®, IMV,
L´Aigle, France ) and it has previously been tested in vitro for freezing
ram semen, with satisfactory results (Gil et al., 2003). A soybean lecithin-
based extender (AndroMed®, Minitüb, Germany) is a Tris-based buffer
containing phospholipids, citric acid, sugars, antioxidant and glycerol,
and has been utilized successfully for cryopreservation of bovine (Aires
et al.,2003) and ovine semen (Fukui et al.,2008). Gil et al. (2003) found
that, the subjective motility was slightly higher in Bioexcell extender than
in the milk extender (47 vs. 46.5%; NS), as was membrane integrity (38
vs. 37.7%; NS), and the percentage of incapacitated spermatozoa
(28.5vs.26.3%; NS). Furthermore, the results obtained indicate that when
freezing ram semen, Bioexcell containing 6.4% glycerol may be used as
31
an alternative extender to the conventional milk extender containing 5%
egg yolk (Gil et al., 2003).
The diluents have a quadratic effect on motility when increasing
glucose concentration in the diluents with a maximum of 55 mM. At this
sugar concentration, motility was higher for diluents containing glucose
and fructose than for containing lactose, sucrose or trehalose (Molinia et
al., 1994).
Semen processing protocols when centrifugation was included,
percentages of motile spermatozoa, sperm with intact membrane as well
as incapacitated spermatozoa were higher compared to those processed
without centrifugation, but the situation was opposite when the sperm
numbers were calculated (Söderquist, 1999). The protocol with adjusted
extension of semen yields higher numbers of viable spermatozoa per A1
dose compared to the protocol involving semen centrifugation (Gil,
1999). However, semen dilution in different ratios (v/v) had no effect,
since the overall post-thaw spermatozoa viability was highly dependant
on the freezing method and the cryoprotectant used (Pontbriand et al.,
1989). Moreover, Colas (1975) reported that, ram semen diluted to a
constant concentration rather than volume to volume achieved superior
results.
32
1.8 Semen freezing:
Polge et al. (1949) were the first to report a protective effect of
glycerol, which in combination with low rates of cooling, resulted in high
survivals after freezing and thawing of spermatozoa from several species.
The challenge to cells during freezing is not their ability to endure storage
at very low temperatures; rather it is the lethal effect of an intermediate
zone of temperature (-15° to -60 °C) that the cell must traverse twice,
once during cooling and once during thawing (Mazur, 1985).
The goal of freezing semen is to decrease the temperature from 5°
to -196°C in gradual steps to avoid damaging the delicate sperm cells.
This can be done by utilizing cryogenic nitrogen vapour, dry ice or
alcohol bath (Herman et al., 1994). The temperature variation involved in
freezing and thawing of semen, however, inevitably reduces the
proportion of motile spermatozoa and causes ultra-structural, biochemical
and functional damage (Söderquist, 1999). Healey (1969) reported that,
the ram frozen semen examined in the electron microscope, showed
consistent damage to the outer spermatozoal membrane and acrosome
complex, the damage ranged from slight swelling of the acrosome to total
removal of cytoplasmic regions. The initial freezing temperature has a
significant effect on spermatozoa motility and velocity following post-
thawing , were the best motile spermatozoa achieved at – 125°C freezing
33
temperature (Bag et al.,2002). However, the freezing in small dimensions
straws resulted in a very quick drop of temperature, leading to
intracellular crystallization of water, this may cause less damage to the
cells during the temperature drop than the extreme dehydration associated
with slow freezing (Berg, 1999). Pontbriand et al. (1989) cited that,
cooling rates of 6 to 24 °C per minute and 10 to 100 °C per minute have
been reported as acceptable, demonstrating that ram spermatozoa tolerate
a wide range of cooling velocities. Kaya et al. (2001) concluded that,
melatonin administration to sperm donors improved freezability of ram
semen collected from these rams and reduced enzyme leakage through
sperm cells during cryopreservation. Although the manual freezing of
straws in a liquid nitrogen vapour 5 cm above the liquid nitrogen in
freezing tank was comparable to that of automatic temperature controlled
liquid nitrogen freezing chambers, the later may provide more reliable
results when cryopreserving semen from rams under field conditions
(Pontbriand et al., 1989).
1.9 Cryopreservation of spermatozoa:
Sperm survival after freezing-thawing is dependent on freezing
technique, cryopreservation, diluents composition, glycerol
concentration, dilution rate, cooling, equilibration interval and thawing
method (Graham et al., 1978; Bearden and Fuquay, 1984).
34
Cryopreservation of spermatozoa for many species leads to series of
alterations resulting in a marked decrease in their fertility (Parks and
Graham, 1992). Freezing and thawing lead to a decrease in both
membrane integrity and acrosome integrity.
It is suggested that the structural integrity of ram spermatozoa is largely
un-affected after cryopreservation, and that damage to plasma membrane
is primarily responsible for the low fertility of frozen–thawed
spermatozoa (Valcarcel et al., 1996). The processing and storage of ram
semen lead to reduced sperm motility and disruption of membrane
integrity. It is generally assumed that these changes are detrimental, and
are associated with reduced fertilizing ability after cervical insemination.
However, recent evidence suggests that, while many spermatozoa remain
motile after storage, the membranes of the motile spermatozoa are
destabilized to the point where they may not survive further ageing in the
female reproductive tract after cervical insemination (Maxwell and
Watson, 1996).These membrane changes are similar to the capacitation of
acrosome reaction of spermatozoa (Maxwell and Watson, 1996;
Chatterjee et al., 2001). Thus, stored spermatozoa may require less
capacitation time in the female reproductive tract, and may readily
fertilize oocytes if placed in their immediate vicinity, as with in vitro
fertilization, tubal or intra-uterine insemination. It is argued that some of
these changes can be prevented or reversed using antioxidants in order to
35
improve fertility following cervical insemination (Maxwell and Watson,
1996; Mara et al., 2005; Bucak et al., 2008; Atessahin et al., 2008). Colas
(1975) reported that, ram semen cryopreserved in straws resulted in
fertility higher than that of semen frozen through pelleting on dry ice.
Even when semen was stored for eight hours in a refrigerator, conception
rates were significantly lower with refrigerated semen than with fresh
semen (Cordova et al., 1989).
1.10 Thawing of frozen semen:
According to general recommendations, thawing of ram semen
should be carried-out quickly and at a relatively high temperature.
However, if thawing is not fast enough, the ice crystals formed will
increase in size before the melting point is reached, thus injuring the
spermatozoal membranes and other cellular structures (Berg, 1999).
Moreover, thawing straws in a water–bath at a higher velocity
(60°C for 8 seconds) had no effect on spermatozoal motility, progressive
status ratings, or acrosomal integrity when compared to a lower rate
(37°C for 20seconds) (Pontbriand et al.,1989). Söderquist (1999)
concluded that sperm can be thawed at 50°C for 9 seconds instead of
70°C for 5 seconds without further reducing sperm motility or membrane
integrity. Thus lower thaw temperature would facilitate a wide spread use
of frozen–thawed ram semen under farm conditions.
36
Melatonin implantation to ram in the breeding season improved
post-thaw sperm viability and intact acrosome rates without influencing
the motility rate, that the post-thaw alkaline phosphates release through
sperm cells was significantly lower in the melatonin-treated group in
comparison with untreated controls (Kaya et al, 2001). However,
individual ejaculates of ram semen can only be considered suitable for
storage and use for insemination if the percentage of forward moving
spermatozoa is not less than 40% upon thawing and 30% after 5– 6 hours
of incubation (Evans and Maxwell, 1987).
1.11 Oestrous synchronization:
Sheep are seasonally polyoestrus; their normal breeding season
depends upon breed and geographic location. The physiological basis of
the breeding season seems to be induced by a rise in the production of
melatonin by the pineal body during the reduction in the day light (Berg,
1999). Ewes are able to monitor changes in the daily photoperiod by the
circadian secretion of melatonin from the pineal gland. Melatonin output
is regulated by photoperiod and elevated concentrations are found in
blood only during the hours of darkness (O`Callaghan, 1994; Rosa and
Bryant, 2003). The length of oestrus cycle is 16-17 days (Monika, 2006).
The oestrous period lasts approximately two days; ovulation takes place
in the second half of the oestrus, 30-36 hours after the onset of the heat
(Sorensen, 1979; Taljaard et al., 1991). Ewes in oestrus do not exhibit
37
sufficiently clear symptoms of their state and it is necessary to use teaser
males for identification (Chemineau et al., 1991). The time in oestrus can,
to a certain extent, be assessed by examination of the cervical mucus
when the speculum is inserted into the vagina in connection with
insemination. In the first 12 hours of the heat the mucus is absolutely
clear, in the next 12 hours increasingly opaque, in the following 12 hours
white or yellow-white and very viscous, and in the last 12 hours smoke
grey and more dry and solid (Berg, 1999 ).
Levels of plasma progesterone at oestrus ranged from 0.1 to 0.5
ng/ml and during the luteal phase from 3 to 6 ng/ml. Levels found during
seasonal anoestrus were within the range of those observed at oestrus. In
animals which ovulated, the plasma progesterone concentration either
remained basal or rose to a lower level <2 ng/ml than that found during
the luteal phase of the cycle (Haresign et al., 1975).
When Targhee and Fin x Targhee ewes were subjected to different
photoperiods, the percentage of exposed ewes lambing was lowest for
natural day length (37%) and highest for 8 light: 16 hours of darkness
(81%). Considering percentage lambing in combination with interval to
lambing the 8L: 16 D treatment proved to be the most effective (Slyter et
al., 1986).
The treatment of anoestrous ewes with medroxy-progesterone
acetate (MAP) priming increased the ovulation rate by increasing the
38
number of follicles that responded to PMSG (Leyva et al., 1998a). FGA
sponges (fluorogestone acetate), containing 30 mg are recommended for
use in ewes in seasonal anoestrus and 40 mg for ewe lambs and ewes in
the breeding season. MAP sponges contain 60 mg and can be used for all
purposes (Chemineau et al., 1991). Moreover, progestagen treatment is
administered for 10 – 12 days in the non-breeding season or 12 – 14 days
during the breeding season in the ewe (Chemineau et al., 1991). Though,
the treatment with MAP sponges does not adequately synchronize oestrus
and ovulation among cyclic ewes due to the difference to follicular
patterns that results, depending on the stage of the cycle at the time of
sponge insertion (Leyva et al., 1998b). An intramuscular injection of
PMSG at the end of progestagen treatment (at sponge removal) in the
ewe, increases follicular growth, the duration of oestrus, ovulation rate
and advances the onset of oestrus (Chemineau et al., 1991). The use of
progestagen sponges plus PMSG for the induction of out-of-season
breeding in ewes is associated with a number of recognized short-
comings. A possible reason for variability in conception rates is the high
incidence of complete embryonic loss or due to fertilization failure in
ewes that are induced to super-ovulate after treatment with a dose of
PMSG (Haresign, 1992). Langford et al. (1983) found that, ewes
inseminated at 55 and 60 hours after sponge's removal, conception rates
(in June and October) were 82 and 87% with PMSG and 18 and 48%
39
without PMSG respectively. The corresponding lambing rates were 60
and 74 with PMSG and 10 and 26% without PMSG.
Furthermore, melatonin (Regulin) can advance the onset of the
breeding season of commercial sheep flocks. This treatment appears to
overcome a number of the short-comings of progestagen sponges plus
PMSG (Haresign, 1992). Treatment with melatonin implant (18 mg) 35
days before inserting progestagen-impregnated sponges (14 days) and
PMSG (500 iu, on the day of sponge removal) showed AI results
significantly higher in lambing rate compared to ewes not implanted with
melatonin (60.4 vs. 32.6%) (Laliotis et al.,1998).
Pre-treatment of seasonally anoestrous ewes with PMSG (750 iu)
or oestradiol benzoate (50 µ g) 24 or 7 hours respectively before a single
injection of synthetic LH-RH (150 µ g) significantly increased the release
of LH compared to that after injection of LH-RH alone (Haresign and
Lamming, 1978).
Fukui and Roberts (1981) found that, the injection of 24 mg PGF2α
(prostaglandin) between day 6 and 12 of the oestrous cycle resulted in a
higher proportion of ewes exhibiting oestrus (92.9%) within 5 days of
treatment as compared to the other two doses (16.6 and 66.6% for 8 and
16 mg PGF2α, respectively).They concluded that the occurrence of
ovulation depended on the dose of PGF2α rather than the stage of breeding
season.
40
Chemineau et al. (1991) concluded that, timing of AI after oestrous
synchronization with a source of progesterone and PMSG, the optimal
timing for a single insemination (semen either liquid state or in deep-
frozen state) is 55±1 hours after sponge removal. In two inseminations,
they can be performed at 50–60 hours after sponge removal. In ewe
lambs, in which oestrus appears earlier than in adult ewes, it is necessary
to inseminate at 50±1 hour after sponge removal of the implant. Manahil
(1999) reported that, the treatment of Sudan Desert sheep with
progestagen intra-vaginal sponges accompanied by PMSG has a merit
resulting in significantly higher pregnancy rate to first insemination than
luprostiol or progestagen alone.
1.12 Insemination of ewes:
The ewe has a miniature cervix structured like that of the cow. The
circular folds are much more distinct and many in number (six or seven).
The intra-vaginal portion extended further and lies closer to the floor than
in the cow (Sorensen, 1979). The gross anatomy of ovine cervix is
described with particular reference to the positioning of annular folds.
The second fold is eccentric to the other concentric folds and presents a
physical barrier to the passage of a straight instrument, a phenomenon
that has prevented the use of deep cervical or uterine artificial
insemination (Thomas and Homer, 1981). Naqvi et al. (2005) found that,
the average number of funnel shaped folds in the cervix of Malpura and
41
Kheri ewe lambs and adult ewes were 3.2±0.19 and 3.4±0.22. However,
the second and third-folds from the os were observed to be eccentric in
both ewe lambs and adult ewes. The cervix measures 1 cm (0.4 inch) in
diameter and 5 cm (2 inches) long (Sorensen, 1979). However, Souza et
al. (1994) found that, in 134 Polwarth and 138 Corriedale ewes, cervix
length averaged was 5.9 and 6.7 cm respectively, cervix diameter was 0.8
cm in both breeds and a cervical flap was found in 52.2% of the cervixes
examined.
Therefore, in sheep, most AI is performed with diluted fresh semen
while only a few ewes are inseminated with frozen semen (Colas, 1983).
Alsayed (1996) found that, AI in Sudan Desert sheep with undiluted
semen gave 90% conception rate, while diluted semen gave 57.1% and
hand mated controls were 91.6%.
However, deep-frozen ram semen is not easy to use, mainly
because of three factors: fertility of deep-frozen semen is about 20
percent lower than that of fresh semen; the required number of
spermatozoa appears to be higher than fresh semen and the technique is
more expensive and more complicated (Colas, 1975). Since insemination
at an optimal time in the heat is essential to achieve an acceptable fertility
results, it is of utmost importance to be able to preserve and store the
semen under optimal conditions (Söderquist, 1999). There are three
methods of semen deposition in sheep:
42
(i) Vaginal insemination.
(ii) Cervical insemination.
(iii) Laparoscopic insemination.
1.12.1 Vaginal insemination:
Vaginal insemination consists of deposition of semen into the
anterior vagina without an attempt to locate the cervix (Evans and
Maxwell, 1987). The poorest results are obtained when semen is left in
the vagina close to the cervix, the so called-shot in the dark method.
Much improved conception rates result from depositing semen about 1
cm into the folds of the cervix (David, 1994). Liquid ram semen diluted
in a milk–based extender and vaginally inseminated once in natural heat,
with a semen dose of 150 million spermatozoa, gave acceptable fertility
results and is to be recommended as the method of choice in Norway
(Heiko et al., 2003).
1.12.2 Cervical insemination:
Cervical insemination involves deposition of semen to a depth of
up to 3 cm into the cervix. If, as in some animals, the cervix will permit
further passage of the pipette, the method can be adopted to become a
non-surgical intra-uterine insemination (Evans and Maxwell, 1987).
Cervical insemination in sheep using frozen semen has not progressed
due to low conception rates (Salamon and Maxwell, 1995). Naqvi et al.
(1998) found that mid-cervical and trans-cervical insemination with
43
frozen semen resulted in similar lambing rates 28.5 and 22.7 percent
respectively, but no lambing was achieved on os-cervical insemination.
For cervical insemination the dose can vary from 0.5 to 1.0 ml and should
contain from 150 to 200 million viable sperms (Herman et al., 1994).
However, Berg (1999) reported that, the volume that can be deposited in
the cervical canal of the ewe is normally limited about 0.2 ml and this
insemination method necessitates a minimum number of spermatozoa of
approximately 200 million and the semen dose contains at least 50%
normal motile spermatozoa after thawing. Smith et al. (1995) found that,
with trans-cervical insemination method conception results obtained were
higher than partial penetration (52%vs.30%). However, the full
penetration rates with chilled semen produced no better results than
cervical insemination (Smith et al., 1995); while partial penetration was
lower (full 68%, partial 58%). However, Westhuysen et al. (1981) found
that, the number of lambs born by female treated with MAP and PMSG
(300 IU) was significantly higher after double than single insemination in
12 and 24 hours after oestrus was observed. Hair sheep ewes were
synchronized using PGF2α and bred by transcervical insemination with
frozen–thawed semen after 48 hours after the second injection; the
conception rate was (8.7%), while in a subsequent trial, ewes were
synchronized with controlled internal drug release (CIDR) devices
containing 300 mg progesterone for 12 days and inseminated 48 hours
44
after CIDR removal , resulting in conception rate of 52.9% (Godfrey et
al., 1999).
The Guelph system for trans-cervical AI (GST-AI), as described by
Halbert et al. (1990 a ) and Halbert et al . (1990 b), provides a viable non-
surgical alternative to laparoscopic AI. The Guelph system allows for
consistent penetration of the offset rings of the ewe`s cervix, thus
permitting the semen to be dispelled directly into the body of the uterus.
In many instances, conception rates using this technique have compared
favourably with those achieved using laparoscopic AI (Cunningham and
Deborah, 1994 ). The efficiency (time, labour, equipment and drugs) of
this technique makes the Guelph system comparable to the laparoscopic
method. The equipment is less expensive and time , labour and materials
are not required to prepare the ewe for surgery and no anaesthesia or
sedation is necessary (Naqvi et al.,1997; Anel et al.,2006). However,
Cambell et al. (1996) found that, varying degrees of damage occurs to
the cervical lining over the length of the cervix when the trans-cervical AI
needle penetrated by use of GST-AI technique. Exogenous oxytocin aids
in the transcervical passage of an AI gun into the uterus of ewes, and it
may be an effective adjunct to sheep AI procedures (Sayre and Lewis,
1996). Recently, Ivanka et al.(2009) found that, the treatment with
Cervdil® (East Kilbride, Scotland), a prostaglandin E2 (PGE2) releasing
45
vaginal insert used for induction of cervical ripening and labour in
women, facilitated transcervical semen deposition in anestrous ewes and
has the promise of a technique to improve transcervical AI in sheep.
It is recommended that, trans-cervical insemination be performed
approximately 42 hours after progestagen pessary removal or
approximately 18 hours after the onset of oestrus (Husein et al1996).
Pregnancy rate of 81 percent have been reported by Halbert et al.(1990 b)
using the Guelph system for trans-cervical insemination. However, using
frozen–thawed semen, the trans-cervical insemination yielded lambing
rates of 50.7 percent for ewes bred during the breeding season (Buckrell
et al., 1994). The difference in pregnancy rate between Suffolk and
Belclare ewes following cervical AI with frozen–thawed semen was due
to sperms traversing the cervix and uterus in a higher proportion of
Belclare than Suffolk ewes, leading to a higher incidence of fertilization
(Fair et al., 2005).
1.12.3 Laparoscopic insemination:
Laparoscopic insemination by-passes the cervix and deposits the
semen directly into the uterine horns. It is a minimally-invasive, minor
surgical procedure. With intrauterine AI, a thousand ewes could be
inseminated seasonally with fresh semen from one ram. Using
intrauterine AI and frozen semen, as many as 25000 ewes could be
46
annually inseminated with semen from one highly fertile ram (David,
1994). Lambing rates to AI using frozen semen were found to be 62 and
67 percent for laparoscopic and trans-cervical insemination respectively.
Showing that, laparoscopic AI was confirmed as the most suitable
technique for insemination using frozen semen in sheep breeding
programme (Cappai et al., 1998). Laparoscopic AI method requires less
semen (20 million sperm). However, this method requires skill and
special equipment (Herman et al., 1994). However, Smith et al. (1995)
reported that, with frozen semen there were no significant differences
between trans-cervical and laparoscopic AI (64.1 vs. 66.7%,
respectively).
1.13 Fertility following AI:
Fertility, the proportion of ewes lambing of all those exposed to the
ram during defined period (usually expressed as a percentage) varies with
breed, season, age, nutritional status, breeding management and farm
conditions (Monika, 2006). Assessment of ram fertility on basis of semen
characteristics was the objective of several studies and was described and
evaluated by Cameron and Fairnie (1984). No single test accurately
predicts fertility of a semen sample. However, the examination of various
semen characteristics can determine fertility potential (Nabil et al., 2006).
It is well known that a correlation exists between certain semen
47
characteristics, such as motility and sperm morphology, and field fertility
results for both fresh and frozen semen (Elliot, 1978).
Many studies have shown that within the same breed, there exists a
wide variation in fertility between rams (Mickelesen et al.,1981).
However, Donovan et al.(2004) found that, pregnancy rate was
significantly influenced by breed of ewe and inseminator; these results
suggest that ewe breed may be a critical determinant of the potential for
exploitation of cervical insemination of frozen-thawed semen in sheep
breeding programmes. Langford and Marcus (1982) found that, the
number of ewes lambing after insemination with 200 million sperms were
similar to those bred after natural service at progestagen induced oestrus.
However, when less than or equal to 100 million spermatozoa were
inseminated, fertility fell markedly and the number of lambs per ewe
inseminated decreased (Langford and Marcus, 1982). The total number of
spermatozoa (80 vs. 200 million) inseminated to Karakul ewes in 12 and
22 hours after the onset of the oestrus did not affect the conception rate ,
but it was lower when spermatozoa where in higher volume of fluid (0.2
vs. 0.5 ml) (Roux and Le-Roux , 1976).
Furthermore, Hackett and Wolynetz (1982) observed that, fertility
was 53% for ewes bred by natural mating, 34% for ewes receiving PMSG
and bred by AI and 9% for those not receiving PMSG and bred by AI.
48
Fertility is very high in all Norwegian breed, about 92.5 percent after AI
with fresh diluted semen, this partially due to the favourable
environmental conditions and low lactation (Berg, 1999).
The fertility results after AI with frozen semen remains
unacceptably low (Graham et al., 1978). In Sweden, Söderquist (1999)
reported that, the average overall fertility with frozen-thawed semen in
synchronized ewes was 39.7%. Pontbriand et al. (1989) suggested that
the poor fertility rates after using frozen-thawed ram semen, likely,
results not only from reduced sperm motility, but also from ultra-
structural damage to sperm integrity. No difference in fertility of frozen –
thawed semen adjusted to the final concentration before packing (200
million sperm) with centrifugation or without centrifugation (30.8% vs.
29.7%) and cervically inseminated during oestrus (Gil et al. 2002). Trials
have been conducted on AI using fresh and frozen semen, but the
conception rate with frozen semen has been considerably lower than
fresh semen, mostly in the range of 30- 60 percent, whereas, for fresh
semen it was 70 percent (Dyrmundsson et al., 1989). Frozen semen from
19 individual rams has been evaluated for fertility, using bovine oocytes
in vitro fertilization procedure. The results showed that, there was a
significant difference between rams ranging from 8.0+2.0 to 89.9+0.4 and
a significant difference between ejaculates from the same ram ranging
49
from 61.5+13.0 to 87.5+3.2 (Smith and Murray, 1996). The collection of
semen in the fall, facilitates successful freezing of semen and AI in the
spring and this could be useful to determine whether the low fertility in
the out of season mating is due to the ram, the ewe, or both (Mickelsen et
al., 1981). Heiko et al.(2004) concluded that, the superior fertility results
achieved for minitubes compared to ministraws when cervical
insemination with 200 million sperm have been carefully evaluated in
relation to the possible application of a more rational semen production
and simplified semen handling at AI, when using mini straws thawed at
35° C for 12 seconds. The biological effects of the inorganic constituents
(Na, Cl, Ca, K and inorganic P), together with magnesium (Mg), in the
semen on semen quality should be considered in the interpretation of the
results obtained in the fertility evaluation of the various ram breeds
(Abdel Rahman et al., 2000).
50
Materials and methods
51
2- Materials and Methods:
2.1 Site of study:
This experiment was conducted from April 1999 to March 2000 at
the National Animal Breeding and Artificial Insemination Centre (Kuku,
Khartoum North) where rams were kept and semen collection, evaluation
and processing were carried-out. Insemination of ewes was applied in six
private farms in Khartoum state.
2.2 Latitude and climate:
In Sudan the year is divided into three seasons, winter (November-
February), summer (March–June) and autumn (July–October). In
Khartoum (15° 36 N, 32` E) the summer is dry and very hot (maximum
temperature between 38° and 44°C). Climatic data of temperature,
relative humidity and rainfall during the period of investigation were
collected from the Meteorology Department (Appendex 1).
2.3 Animals:
Six selected Hamari and three selected Kabashi breeding rams
(both are ecotypes of Desert sheep) were used in this study. Rams were
two to four years of age at the beginning of this experiment. Rams were
maintained under uniform conditions of feeding and management. During
the whole period of study, it was observed that, all rams from the two
breeds were free of palpable abnormalities or clinical diseases.
52
2.3.1 Housing:
Each of the rams was housed in an individual shaded pen (2 x 1.6
meters) with enough space for free movement.
2.3.2 Feeding:
Rams were fed with pea-nut hay (ad libitum). Each ram was
supplied daily with 0.5 kg commercial concentrate (Al Gandol Feed) with
free access to water and mineral licks (10 kg).
The chemical analysis of concentrate feed on dry matter basis was
as follows:
Metabolic energy 11481.39 M.J.
Crude protein 16.5 %
Crude fibres 10 %
Plus different vitamins and minerals needed by the animal.
The mineral blocks composed of (per Kg):
Sodium 37.1 % Zinc 2.5 mg
Magnesium 3.5 mg Iodine 38 mg
Iron 3.0 mg Cobalt 35 mg
Manganese 625 mg Selenium 13 mg
53
2.3.3 Body weight recording:
The rams were weighed once monthly and body weight was
recorded using a weighing scale.
2.3.4 Scrotal measurements:
Scrotal circumference (SC) and testicular length measurements
were taken while the ram was in the standing position. The testicles were
pushed into the bottom of the scrotum and measured around the widest
point of the testicles with a flexible measuring nylon tape (Mickelsen et
al., 1981).
2.4 Reaction time (RT):
Sexual drive of each ram was estimated by measuring the reaction
time which denotes the interval between the first contacts with the teaser
ewe until ejaculation is completed (Ibrahim, 1997).
2.5 Semen collection:
Rams were subjected to a training period of two weeks to ejaculate
into an artificial vagina (AV) after mounting anoestrous ewe which was
restrained and tied to a post or held by an assistant.
Semen was collected from all rams by means of AV (IMV model
for ovine). This was continued for one year covering the three seasons
(Summer, autumn and winter).
54
A total of 293 first ejaculates of semen were collected during the
whole period of the study, of which 71 and 22 ejaculates were collected
during the summer, 42 and 24 ejaculates were collected during the
autumn and 85 and 49 ejaculates were collected during the winter from
Hamari and Kabashi rams respectively.
A separate AV was used for one collection only. The AV case was
half-filled with warm water (48-50°C) through a valve fitted in one side.
One end of the inner liner was lightly lubricated with sterile Vaseline to a
depth of three centimetres. The AV was inflated with air through the
valve, this exerts some pressure in the AV cavity but allows easy
penetration of the penis. The temperature of the AV just before semen
collection was around 42-44°C and checked by insertion of a clean
thermometer.
The teaser ewe was secured and tied to a post or held by an
assistant. The operator took a crouching or kneeling position at the right
side of the teaser, and held the AV in the right hand along the flank, with
the open end facing towards the ram and down-wards at an angle of 45°.
The valve of AV was directed down-wards to avoid contact with the ram.
When the ram mounted, the erected penis was directed into the
open end of the AV, the left hand was used to grasp gently the sheath and
deflect the penis into the open end of the AV.
55
A vigorous up-ward and forward thrust indicated the occurrence of
ejaculation.
Immediately after collection, the AV was turned up while keeping
collecting glass up-right, the pressure was released from the inner liner by
opening the valve for water and air to escape.
2.6 Semen evaluation :
All procedures of semen evaluations were carried-out at the semen
laboratory, immediately after collection as described by Rao (1971).
2.6.1 Appearance of ejaculate:
Appearance of semen was estimated directly from the collection
tube for contaminants such as hair, dirt, faeces, blood and urine.
2.6.2 Volume of ejaculate:
The volume of the ejaculate was measured directly from the
graduated collecting glass tube in millilitres (ml) and to the nearest 0.1 ml
2.6.3 Colour and consistency of semen:
Colour and consistency of semen were assessed by visual
observation directly in the collecting glass tube.
56
2.6.4 Sperm motility:
2.6.4.1 Mass activity (Gross motility):
Semen mass activity was estimated by observing the degree of
wave motion and general activity of spermatozoa. Mass activity was
estimated by examining a drop of fresh semen on a pre-warmed slide
(37°C) under light microscopy at magnification (10 x objective). The
mass activity is graded from zero to 5 scores according to intensity of
wave motion as described by Evans and Maxwell (1987). The basis of
this method includes:
0 = (Dead) all spermatozoa are motionless
1 = (Very poor) very few spermatozoa (about 10%) are
active, with weak movement only.
2 = (Poor) no waves are formed, but some movement of
spermatozoa are visible . Only 20-40% of sperm cells are
active, and their motility is poor.
3 = (Fair) only small , slow moving waves. Individual
spermatozoa can be observed . 45-65% of sperm cells
are active .
4 = (Good) vigorous movement, but the waves are not so
rapid as for score 5. About 70-85% of sperm cells are active.
57
5 = (Very good) dense, very rapidly moving waves. Individual
Sperm cells can not be observed. 90% or more of the
spermatozoa are active.
2.6.4.2 Individual motility:
Individual motility of sperm was estimated by examining a drop of
diluted semen with physiological saline solution (0.9% Na Cl) on a pre-
warmed slide (37°C). One drop of semen and two drops of normal saline
were mixed together and covered with a cover-slip. Under a phase
contrast microscope fitted with a hot stage maintained at 37°C and at
magnification (400 x), the individual motility was estimated at a scale
ranging from zero to 100 according to the percentage of sperms moving
straight forward over the field of the microscope. Only sperm that have
forward or progressive movements are included, but sperm with
backward, vibrating or circling movements are not included (Evans and
Maxwell, 1987).
2.6.5 Sperm cell concentration:
The concentration of spermatozoa was counted by the aid of a
haemocytometer according to the method described by Evans and
Maxwell (1987). An improved Neübauer chamber was used. With the red
blood sampler pipette (that has the red agitator in the bulb) a sample of
raw semen was sucked up to the 0.5 mark. The distilled water was drawn
58
up to the 101 mark of the pipette (made 1/200 dilution with distilled
water). The pipette was agitated by holding it between the thumb and the
index finger and was shaking to ensure dilution of semen sample. Four to
five (4–5) drops were discarded from the end of the pipette to obtain
properly diluted semen from the bulb. The diluted semen with the tip of
the pipette was allowed to run under the cover–glass of the
haemocytometer, then the spermatozoa were allowed to settle for 5–6
minutes before counting. Five large squares, one at each corner and the
one in the centre were counted. Spermatozoa heads lied on the top and
right lines between the squares were included in the counting of each
large or small square.
The spermatozoa in the large squares (5 x 16 = 80 small squares)
were counted to determine the sperm concentration by applying the
following formula:
The number of sperm counted over 80 small squares x 5 (because
only one-fifth of the total small squares were counted) x 10,000 (to
convert the number of sperm counted in the 0.1 cu mm volume to a
millilitre) x the dilution factor.
Number of sperms per millilitre =
No. of sperms counted x 5 x 200 x10 x 1000
= No. of sperms counted x 107
59
2.6.6 Sperm cell morphology:
2.6.6.1 Wet preparation:
The semen sample was fixed in buffer formal saline in a ratio of
1:10 immediately after collection. A drop of diluted semen was placed on
a microscope slide under cover-slip. The preparation was left for a couple
of minutes to allow sperm cells to settle down. Under phase-contrast
microscopy at magnification of (400x), two hundred sperms were counted
for abnormalities, loose heads, proximal cytoplasmic droplets, mid-piece
and tail abnormalities. The results were expressed as a percentage for
each abnormality in different ejaculates (Rao, 1971).
2.6.6.2 Dry preparation:
Sperm head and acrosome abnormalities were evaluated on air-
dried eosin-nigrosin stained slide. Two hundred spermatozoa were
examined on each slide under oil immersion lens at magnification of (400
x) and incidence of each category was expressed as a percentage for head
and acrosome abnormalities in each ejaculate (Evans and Maxwell,
1987).
2.6.7 Live-dead spermatozoa:
Eosin-nigrosin was used for routine examination of live-dead
spermatozoa. A small drop of semen was mixed well with two drops of
60
the stain on a pre-warmed slide (37°C). The drops were allowed to reach
body temperature before mixing. Thin smear was prepared from the
mixture. Under high power magnification (400x), two hundred
spermatozoa were examined for live and dead cells. Sperm heads or at
least two thirds of the head stained red, were considered as dead
spermatozoa. The result was expressed as a percentage for live-dead
sperms in each ejaculate (Rao, 1971).
2.7 Extender preparation:
The extender was prepared a day before collection. Tris-based
diluent used in this study was composed of a solution of 3.634 gram (gm)
Tri-hydroxy methyl amino methane (Tris), 0.5 (gm) glucose, 1.99 (gm)
citric acid monohydrate, 15 ml egg yolk, 5 ml glycerol, 100000 i.u
penicillin, 100 mg streptomycin and distilled water added to the level of
100 ml (Evans and Maxwell, 1987).
2.8 Semen dilution:
Dilution of fresh semen was done within the first 10–15 minutes
following collection and evaluation. The ejaculate was placed in a water
bath at 37°C and the diluent was at the same temperature when mixed
with the semen. Depending on a dilution rate (1 semen: 4 diluent), only
good samples of semen were diluted. A clean glass pipette was filled with
required amount of extender, and slowly added to the semen in one–step
61
extension methodology. Then the diluted semen was mixed gently and
was re-examined for sperm motility (Evans and Maxwell, 1987).
2.9 Semen cooling:
The diluted semen in the flask was carried in a portable water bath
at 32°C. The water level should be higher than that of the diluted semen
inside the flask. Then the water-bath with diluted semen in the flask was
placed inside the upper part of a cold cabinet at 5°C. When temperature
of diluted semen reached 20°C, ice-cubes were added to the water-bath
every five minutes to maintain an even drop in temperature to 5°C.
However, the temperature of diluted semen was reduced form 32°C to
5°C within 45 to 60 minutes, depending on the volume of diluted semen.
Then the diluted semen was left inside the cold cabinet for another one
hour before packing.
2.10 Semen Packing:
Medium size (0.5 ml) IMV plastic straws were filled with the
diluted semen by suction. Each straw contained 200x106 spermatozoa.
The air space at the end of the straws was created by a plastic comb
before sealing with the polyvinyl alcohol powder to prevent straws crack
during freezing. The plugged straws were immersed for two hours in a
water–bath at 5°C to allow equilibration of sperm with diluent
components and to let the plugs to get harder and the excess powder on
62
the end of the straws fall to the bottom of the bath. Then the straws were
carefully dried with a pre-cold linen towel inside the cold cabinet.
2.11 semen Freezing:
The filled straws were placed horizontally on a cold rack at 5°C
and then lowered into liquid nitrogen vapour LN2 (-80°C) 3-4 centimetres
above the surface of liquid nitrogen in a styrofoam container for 8
minutes. Then the frozen straws were plunged in liquid nitrogen (-196°C)
and then transferred to a storage container.
2.12 Pre- and post-freezing rejected samples:
Diluted semen that showed less than 60% progressive motility after
cooling to 5°C and just before freezing was discarded and considered as a
pre-rejected sample and that frozen semen gave less than 50 percent post-
thawing motility was discarded and considered as a post-rejected sample.
2.13 Thawing of frozen semen:
From each frozen semen batch, two straws, which had been frozen
and stored in liquid nitrogen for a minimum of 24 h, were thawed by
immersion in warmed water at 37°C for 20 seconds (Evans and Maxwell,
1987). Immediately after thawing of semen, the examination of motility
was done on a pre-warmed slide (37°C) with a cover-slip, under phase
contrast microscope. Individual ejaculates of ram semen can only be
considered suitable for storage and use for insemination when the
63
percentage of forward moving spermatozoa was not less than 50 percent
post- thawing (Berg, 1999).
2.14 Oestrous synchronization:
Ewes were treated with progestagen impregnated intra-vaginal
sponges (Chrono-gest®sponges- Intervet) for 14 days and were injected
with 500 i.u PMSG (pregnant mare serum gonadotrophin - Folligon®-
Intervet) intra-muscular at the time of sponge's removal.
2.15 Insemination of ewes:
A total of 114 first inseminations were performed. Of these, 63
inseminations were done with Kabashi and 51 with Hamari frozen-
thawed semen. All females were inseminated in the cervix with 0.5 ml
frozen- thawed semen at 55 hour after sponge removal. The ewes were
presented for cervical insemination by an assistant lifting the hind
quarters of the ewe over, while the front legs remained standing on the
ground. The steps for cervical insemination were as follows:
(1) A speculum was introduced carefully in the vagina of the ewe (10–
13 cm) and located the cervix.
(2) The amount of the mucus observed in the vagina was drained when
it was necessary.
64
(3) The insemination syringe was introduced as far as possible into the
cervix. Twisting motion but with no force with the syringe was
used.
(4) Then the plunger of the AI syringe was pressed to expel the semen
while the syringe was drawn slightly (Evans and Maxwell, 1987).
2.16 Conception rate:
The pregnancy was detected by palpation after three months of
insemination. The reproductive parameters used in this study were
fertility and prolificacy. The fertility is the proportion of ewes lambing of
all those artificially inseminated, whereas, the prolificacy is the number
of lambs born per lambing ewe (usually are expressed as a percentage).
2.17 Experimental design:
• Experiment (1) was design to study the influence of
season and breed on reaction time (RT); semen
characteristic, semen volume, sperm motility, sperm cell
concentration, sperm cell abnormalities and live sperm
cells of semen collecte from 6 Hamari and 3 Kabashi
rams. The relationship between ejaculate volume, sperm
cell concentration, body weight and scrotal
circumference were included. The methodology was
described in materials and methods.
65
• Experiment (2) was design to study the influence of
breed and seasons on freezability of semen obtained
from the two breeds, consisted of the post-thaw
evaluation of frozen ejaculates. Two random straws from
each batch were used to assess the progressive motility
of semen as described in materials and methods.
• Experiment (3) to study the conception rate of oestrous
synchronized ewes (114) in different 6 farms
inseminated with frozen semen obtained from the two
breeds according to the protocol shown in materials and
methods.
2.18 Statistical analysis:
Data were treated and analysed statistically using SPSS 10.1
Package (Statistical Products and Service Solutions for windows) (SPSS
inc., 1999). Physiological and morphological results of semen, post
thawing motility and reaction time were represented as mean+standard
error of the mean. The data were subjected to analysis of variance using
the GLM (General linear Model) and mixed model procedure. The
statistical model included the effect of breed (2), season (3) and
breed*season interaction.
Yijk = µ + βi + Sj + ( βS)ij + eijk
66
Where:
Yijk = Trait measure on individual kth ram.
µ = Over all mean.
βi = The effect of the ith breed.
i = 1,2.
Sj = Effect of the jth season.
j = 1,3.
(βS)ij = interaction between breedi and seasonj .
eijk = Random error term associated with single records.
The testing of differences between means was done using Scheffe`s
multiple range test.
Correlation analysis using Pearson correlation procedure of SPSS
was used to assess the relationship between body weight, scrotal
circumference, semen volume and sperm cell concentration.
The significance of the difference between the two breeds in
pregnancy rate (number of pregnant ewes/number of ewe inseminated
X100%) was tested using the chi-square test. Prolificacy (number of
lambs born/number of ewes lambed) in the two breeds was compared
using the Student`s t- test. Values of p<0.05 was chosen as an indication
of significance.
67
Results
68
3-Results:
3-1 Semen characteristics:
3-1-1 Colour and consistency of ejaculate:
The colour and consistency of most semen ejaculates were white-
creamy (70.20 and 58.95%), white-milky (22.22 and 31.58%) and watery
(7.58 and 9.47%) for Hamari and Kabashi rams respectively (Table 1).
Statistical analysis showed that, breed had no effect on semen colour and
consistency, but winter exerted a significant effect on this character. The
watery ejaculates increased during summer and decreased during winter
(12.73 and 18.18 vs. 3.53 and 4.08%) for Hamari and Kabashi rams
respectively (Table 2).
Table (1) Number and percentage of ejaculates with different colour
and consistency of Hamari and Kabashi rams (mean±S.E).
Parameter Hamari Kabashi
White-creamy 139 (70.2%) 56 (58.95%)
White-milky 44 (22.22%) 30 (31.58%)
Watery 15 (7.58%) 9 (9.47%)
69
Table (2) Number and percentage of watery ejaculates in
different seasons of the year of Hamari and Kabashi rams
Season Hamari Kabashi
Summer 9 (12.68)a 4 (18.18)a
Autumn 3 (7.1)a 3 (12.5)a
Winter 3 (3.53)b 2 (4.08)b
a, b, different superscripts in the same column indicate significant differences (P<0.05).
3-1-2 Ejaculate volume :
The results in table (3) indicated that the mean ejaculate volume for
Hamari and Kabashi rams were 1.31±0.04 and 1.23±0.0.06 ml
respectively. The highest ejaculate volumes (2.5 and 2.3 ml) were
collected during autumn and the lowest ejaculate volumes (0.5 and 0.6
ml) were collected during summer from Hamari and Kabashi rams
respectively. No difference in the mean ejaculate volume was found in
this study between both breeds and seasons. Breed and season of the year
did not exert any significant effect on this semen trait (Table 3&4).
70
Table (3): Means and standard errors of semen traits in different breeds (M±S.E.)
Parameters Hamari Kabashi
Ejaculate volume (ml) 1.31±0.04 1.23±0.06
Mass motility (1-5 scores) 2.73±0.06 2.37±0.08
Motility ( % ) 68.11±0.79 67.91±1.10
Sperm concentration (x 106/ml) 2.83±0.06 2.95±0.08
Live sperm ( % ) 76.90±1.25a 84.73±1.93b
Total abnormalities( % ) 4.26±1.01 4.47±1.18
a, b, different superscripts in the same row indicate significant differences ( P<0.05).
3-1-3 Mass activity:
As in tables (3 and 4) mean score for mass activity were
2.73±0.06 and 2.37±0.08 for Hamari and kabashi rams respectively.
The highest mean mass activity were recorded during winter
(3.16±0.07 and 3.47±0.10), while the lowest mean scores were
recorded during summer (2.48±0.08 and 2.32±0.15) for Hamari and
Kabashi breed-type respectively. Statistical analysis revealed no
breed effect on mass activity but winter season had a highly
significant effect (P< 0.01) on this trait. The interaction term between
breeds and seasons was significant. This indicates that the ranking of
the two breeds varied among seasons.
71
Table (4): Means and standard errors of semen traits in different seasons (M±S.E.)
Summer Autumn Winter Parameter
Hamari Kabashi Hamari Kabashi Hamari Kabashi
Ejaculate volume(ml )
1.33±0.06 1.18±0.10 1.36±0.09 1.29±0.10 1.23±0.05 1.21±0.07
Mass motility (1-5 scores)
2.48±0.08a 2.32±0.15a 2.56±0.13a 2.42±0.15a 3.16±0.07b 3.47±0.10b
Motility (% )
60.78±1.19a 60.46±2.14a 71.88±1.78b 71.04±2.05b 71.68±1.03b 72.25±1.44b
Sperm concentration
(x106/ml)
2.39±0.09a 2.52±0.15a 2.88±0.13b 3.15± 0.15b 3.21± 0.07b 3.18±0.10b
Live sperm (%)
74.20±2.22a 82.80±3.84a 77.58±1.98b 85.25±3.04b 78.93±2.30b 86.13±3.04b
Total abnormalities
( % )
5.16±1.20a 5.40±1.51a 3.97±1.02b 4.23±1.12b 3.68±0.84b 3.78±0.97b
a,b, different superscripts in the same row indicate significant differences ( P< 0.05).
3-1-4 Individual motility:
Results furnished in tables (3 and 4) showed that the average
individual motility were 68.11±0.79 and 67.91±1.10 percent for Hamari
and kabashi breeds respectively .The highest mean motility were found to
be 71.88±1.78 and 72.25±1.44 percent during autumn and winter,
whereas, the lowest mean motility were 60.78±1.19 and 60.46±2.14
72
percent during summer for Hamari and Kabashi breeds respectively.
Summer season showed a highly significant effect (P< 0.01) on
individual motility but breed had no such effect.
3-1-5 Sperm cell concentration:
Results of spermatozoa count presented in tables (3 and 4) showed
that the average sperm cell concentration per ml for Hamari and Kabashi
rams semen were 2.83±0.06 and 2.95±0.08(x109) respectively. The
highest mean sperm concentrations were counted during winter
(3.21±0.07 and 3.18±0.10 billion spermatozoa/ml) while the lowest mean
concentrations were counted during summer (2.39±0.09 and 2.52±0.15
billion spermatozoa/ml) for Hamari and Kabashi breeds respectively.
Statistical analysis showed that breed and summer season exerted a
highly significant effect (P< 0.01) on this trait.
3-1-6 Live sperm count:
The mean live sperm cell percentage in this study was
found to be 76.90±1.25 and 84.73±1.93 percent for Hamari and
Kabashi rams respectively (Table 3). The average live sperm cell
percentage was higher during autumn and winter (78.93± 2.30 and
85.25±3.04 percent) and the lowest values were observed during
summer (74.20±2.22 and 82.80±3.84 percent) for Hamari and
Kabashi rams respectively. Breed and summer season exerted a
73
highly significant effect (P< 0.01) on this character. The interaction
term between breeds and seasons was highly significant. This
indicates that the ranking of the two breeds varied among seasons
(Table 4).
3-1-7 Sperm cell abnormalities:
The results in tables (4, 5 & 6) showed that the mean percentage
for mid-piece, tail, head, acrosome abnormalities, loose heads and
total abnormalities were 1.91±0.14 and 1.52±0.16; 5.42±0.21 and
5.95± 0.31; 6.38±0.21 and 6.65±0.32; 1.73±0.12 and 1.64± 0.18;
5.88±0.19 and 6.57±0.29; 4.26±1.01 and 4.47±1.18 for Hamari and
Kabashi rams respectively. The incidences of mid-piece and tail
abnormalities were significantly affected by summer season (P< 0.05)
where, head abnormalities and loose heads were highly affected by
summer season (p<0.01). Breed affected significantly (p<0.05) mid-
piece abnormalities. There was a significant difference between
summer and autumn on acrosome abnormalities. However, winter
was intermediate and it was not significantly different from either
summer or autumn. The mean percentage of total abnormalities were
higher (5.16±1.20 and 5.40±1.51) in summer than winter (3.68±0.84
and 3.78±0.97) for Hamari and Kabashi breeds respectively.
74
3-2 Live body weight:
The initial average of live body weights at the start of the
experiment were 71.60±1.91 and 60.50±2.50 kg, whereas, the
final weights at the end of the experiment were 81.78±2.06 and
71.13±2.73 kg for Hamari and Kabashi rams respectively. Season
of the year did not exert a significant effect on live body weight.
Table (5): Means and standard errors of sperm cell abnormalities in different breeds (M±S.E.)
Parameters Hamari Kabashi
Mid-piece
Abnormalities
1.91±0.14a 1.52±0.16b
Tail
Abnormalities
5.42±0.21 5.95±0.31
Head
Abnormalities
6.38±0.21 6.65±0.32
Acrosome
Abnormalities
1.73±0.12 1.64±0.18
Loose heads
5.88±0.19 6.57±0.29
a,b, different superscripts in the same row indicate significant differences ( P< 0.05).
75
3-3 Scrotal circumference:
The average scrotal circumference measurements in this study
were found to be 33.12±0.21 and 31.38±0.31 cm for Hamari and
Kabashi rams respectively (table 7). Breed showed a highly
significant effect (p<0.01) on scrotal circumference measurements
but the season of the year did not. The interaction term between
breeds and seasons was significant. This indicates that the ranking of
the two breeds varied among seasons.
Table (6): Means and standard errors of sperm cell abnormalities in different seasons (M±S.E.)
Summer Autumn Winter Parameters
Hamari Kabashi Hamari Kabashi Hamari Kabashi
Mid-piece
Abnormalities
2.33±0.18a 1.80±0.31a 1.77±0.17b 1.38±0.25b 1.64±0.19b 1.38±0.25b
Tail
Abnormalities
6.4±0.37a 6.60±0.62a 4.71±0.34b 6.00±0.49b 5.14±0.37b 5.25±0.49b
Head
Abnormalities
7.80±0.37a 8.20±0.65a 6.41±0.35b 5.75±0.51b 4.93±0.39b 6.00±0.51b
Acrosome
Abnormalities
2.20±0.21a 1.80±0.36a 1.35±0.20b 1.63±0.29b 1.64±0.22ab 1.50±0.29ab
Loose Heads
7.07±0.34a 8.60±0.59a 5.59±0.32b 6.38±0.46b 5.07±0.35b 4.75±0.46b
a ,b, different superscripts in the same row indicate significant differences ( P< 0.05)
76
3.4 Relationship between body weight, scrotal circumference
semen volme and sperm cell concentration:
The correlations of body weight with scrotal circumference were
found to be 0.31 (weak) and 0.70 (strong) and with semen volume were
0.90 (strong) and 0.28 (weak) and with sperm cell concentration were
0.11 (weak) and 0.01 (weak) for Hamari and Kabashi rams respectively
(table 8 & 9).
(Figure 1) The effect of season on sperm abnormalities of Hamari rams.
77
(Figure 2) The effect of season on sperm abnormalities of Kabashi rams.
3.5 Reaction time (RT):
Results illustrated in table (10) showed that the average RTs were
16.41±0.46 and 20.10±1.10 seconds for Hamari and Kabashi rams
respectively. The shortest RTs were recorded in summer and autumn
(14 seconds) in both breed-types, whereas, the longest RTs were
recorded in winter (45 seconds) for Hamari and (35 seconds) for
Kabashi rams. The breed and winter season exerted a highly significant
effect on RT (P< 0.05).
78
(Figure 3) The effect of season on pre- and post rejection sample rate of Hamari rams.
(Figure 4) The effect of season on pre- and post rejection sample rate of Kabashi.
79
3.6 Freezability of spermatozoa :
The mean values of freezability of sperms were expressed in terms
of percent post-thaw motility. In the present study the values obtained
were 56.75±5.95 and 56.19±5.32 percent (table 11). Regarding sperm
freezability, neither the breed nor the season was affecting the trait
significantly. The rejected diluted ejaculates of semen before and after
freezing were 30.77%, 32.44% and 15.38%, 6.35% for Hamari and
Kabashi rams respectively. Breed and winter season exerted significant
effect on post-freezing rejection rate (P< 0.05) in both breeds with better
results obtained of Kabashi rams semen.
Table (7):Effect of season and breed on scrotal circumference (Sc) (cm) and body weight (B.wt) (Kg) (mean±S.E)
Breed
Hamari Kabashi
Parameters
B.wt. Sc. B.wt. Sc.
Summer
73.60±1.99 32.63±0.36N.S 61.20±3.45 31.00±0.63N.S
Autumn
75.05±1.87 33.45±0.32N.S 63.00±2.73 31.38±0.50N.S
Winter
81.78±2.06 33.29±0.37N.S 71.13±2.73 31.75±0.50N.S
over-all mean
76.82±1.14 33.12±0.21 65.11±1.73 31.38±0.31
Significance difference at p< 0.05
80
Table (8): Person`s correlation coefficient between body weight (B.wt), scrotal circumference (Sc), semen volume (SV) and sperm cell concentration (SCC)
from Hamari rams.
Correlation significance * P< 0.05 ** P< 0.01
Table (9): Person’s correlation coefficient between body weight (B.wt), scrotal circumference (Sc), semen volume(SV) and sperm cell concentration (SCC) from
Kabashi rams.
Parameters
B.wt.
SC
SV
SCC
B.wt
1
SC
0.70∗
1
SV
0.28
0.38
1
SCC
0.01
0.01
0.08
1
Correlation significance * P< 0.05
Parameters
B.wt. Sc Sv Scc
B.wt.
1
Sc
0.31
1
Sv
0.90*
0.18
1
Scc
0.11
0.54**
0.28
1
81
Table (10): The reaction time ( RT ) of Hamari and Kabashi rams in different seasons of the year ( mean±S.E)
Season
Hamari
Kabashi
Summer
18.33±0.80a
24.80±1.39a
Autumn
17.47±0.75a
19.13±1.10a
Winter
13.43±0.83b
16.38±1.10b
over-all mean
16.41±0.46
20.10±1.10
a ,b ,c, different superscripts in the same column indicate significant differences ( P< 0.05).
3.7 Conception rate:
During the pregnancy period there was only one case of early
abortion but no incidence of stillbirth or mortality of pregnant ewes or
any other physical abnormalities were detected.
As shown in table (12), the pregnancy rate was (35.3% and 41.3%) and
lambing rate was (35.3% and 39.7%). Chi-square test of the cross-
tabulated variables indicated no significant differences between the
various subclasses. Prolificacy (1.38 and 1.36) was not significantly
different between two breeds.
82
Table (11): The effect of breed and season on the pre- and post-freezing rejection batches and post-motilityof frozen semen.
Pre-freezing rejection (%)
Post-freezing rejection (%)
Post-thawing motility (%)
Season
Hamari Kabashi Hamari Kabashi Hamari Kabashi Summer
32.60a 31.00a 24.57a 19.00a 56.96±
6.48 59.00±
5.83 Autumn
33.33a 50.00a 5.56a 00.00b 55.00±
6.32 55.45±
4.98 Winter
21.43b 16.67b 9.29a 00.00b 57.22±
4.15 55.00±
4.47 over-all mean
30.77 32.44 15.38 6.35 56.75± 5.95
56.19± 5.32
a ,b , different superscripts in the same column indicate significant differences ( P< 0.05).
Table (12): Fertility parameters of frozen-thawed semen obtained from Hamari and Kabashi rams.
Ram breed
Parameters
Kabashi
Hamari
63
51
No. of ewes inseminated
26
(41.3%)N.S
18
(35.3%)N.S
Pregnancy rate (%)
25
(39.7%)N.S
18
(35.3%)N.S
Lambing rate (%)
34
(1.36±0.5)N.S
25
(1.38±0.5)N.S
Prolificacy(Mean±S.E)
Significance difference at p< 0.05
83
(Figure 5) The effect of breed on pre-, post- and total percentage of rejected samples.
84
Discussion
85
4-1 Semen characteristics:
4-1-1 Colour and consistency of ejaculate:
Records of the colour and consistency of the semen samples
observed in the present study indicated that 70.20, 58.95 percent were
white-creamy, 22.22, 31.58 percent white-milky and 7.58 and 9.47
percent watery for Hamari and Kabashi rams respectively. This result
agreed with other results in literature for Sudan Desert sheep (Galil and
Galil, 1982; Alsayed, 1996; Manahil, 1999, Alsayed,2001) and Dabas et
al. (1997) in Patan-wadi rams. The watery ejaculates increased during
summer and decreased during winter (12.68 and 18.18 vs. 3.53 and
4.08% for Hamari and Kabashi rams respectively) and this was due to the
high ambient temperature during summer which affect all the component
of the reproductive function, particularly the quality of semen (Colas,
1983).
4-1-2 Ejaculate volume:
The average volumes of ejaculate obtained in the present study
were 1.31±0.04 and 1.23±0.06 ml for Hamari and Kabashi rams
respectively. They conformed well to those obtained by Alsayed (1996,
2001) in the Sudan Desert sheep. Similarly Daader et al. (1987) reported
1.26±0.01 ml with Rahmani x Finnish Landrace rams in sub-tropical
86
conditions, Williams et al. (1990) reported 1.20 ml with Suffolk rams,
Sackmann and Schone (1990) reported 1.20 ml with Mouflon rams,
Salhab et al. (2003) reported 1.20 ± 0.5 ml with Awassi ram and Kafi et
al. (2004) reported 1.2±0.3 ml with Karakul rams. Slightly higher values
were recorded (1.62 ml) by Manahil (1999) in Sudan Desert sheep.
Lower values of ejaculate volumes were reported by various authors,
Chiboka (1980) with West African Dwarf rams, Galil and Galil (1982)
with Sudan Desert rams , Loubser and Van Niekerk (1983) with Angora
rams, Kalous and Samkova (1991) with Sumava rams, Demirci (1993)
with Awassi rams, Zhang et al. (1995) with Xinjiang Fine wool rams,
Ibrahim (1997) with Local Emirate and Local x Chios rams, Dabas et al.
(1997) with Patan-wadi rams, Rege et al. (2000) with Horro and Menz
rams and Lezama et al. (2003) with Katahdin yearling rams. The non-
significant influence of breed and season of the year on the ejaculate
volume is in agreement with findings of many authors (Vijil, 1987; with
Manchega rams, Daader et al.,1987; with Rahmani x Finnish Landrace
rams and Fernandez, 1993; with Corriedale, Polwarth, Merilin and
Merino rams) and is in disagreement with findings of Brewer et al.
(1995) with Dorsal x Romney rams, Gündogan and Demirci (2003) and
Gündogan (2006); who reported significant dependency of volume of ram
ejaculate on season of the year. Generally semen volumes in both breeds
were within the range of ram semen volume reported in the literature.
87
4-1-3 Mass activity:
The average values obtained for mass activity in the present
study were 2.73±0.06 and 2.37±0.08 for Hamari and Kabashi rams
respectively. These values were lower than those reported by other
authors (Loubser and Van Niekerk, 1983, in Angora rams; Alsayed,
1996, Manahil, 1999, Alsayed, 2001, in Sudan Desert sheep; Kafi et
al., 2004, in Karakul rams). Similarly significant effect of season on
mass activity of spermatozoa was observed by Dufour et al. (1984) in
DLS and Suffolk rams; Vijil (1987) in Manchega rams; Fernandez
(1993) in Corriedale, Polwarth, Merilin and Merino rams; Dabas et
al.(1997) in Patan-wadi rams; Alsayed (2001) in Sudan Desert sheep
and Kafi et al. (2004) in Karakul rams. The interaction term between
breeds and seasons was significant. This indicates that the ranking of
the two breeds varied among seasons.
4-1-4 Individual motility:
The average values of individual motility in this experiment
were 68.11±0.79 and 67.91±1.10 percent for Hamari and Kabashi rams
respectively. These were lower than those values reported by Daader et
al. (1987) in Rahmani x Finnish Landrace rams, Demirci (1993) in
Awassi rams, Zhang et al. (1995) in Xinjiang Fine Wool rams, Manahil
(1999), Alsayed (2001) in Sudan Desert sheep and Salhab et al .(2003) in
88
Awassi rams. However, they were higher than those values recorded in
West African Dwarf rams (Chiboka, 1980), Danube Fine Wool and Dairy
rams (Karcheva and Baulov, 1989) and Sumava rams (Kalous and
Samkova, 1991). The individual motility was highly significantly (p<
0.01) affected by summer season. Similarly significant effect of season on
individual motility were reported by many authors (Dufour et al.,1984,
Vijil, 1987, Daader et al., 1987, Sackmann and Schone ,1990, Alsayed,
2001, Gündogan and Demirci ,2003 and Gündogan, 2006).
4-1-5 Sperm cell concentration:
The average values of sperm cell count per ml in the present
study were 2.83±0.06 and 2.95±0.08 billion spermatozoa for Hamari and
Kabashi rams respectively. They were similar to those results in the
literature of Sudan Desert sheep (Galil and Galil, 1982; Alsayed, 1996
and 2001; and Dabas et al., 1997). Sperm cell concentration in this study
were relatively lower than those recorded by Demirci (1993); Ibrahim
(1997); Manahil (1999) and Salhab et al. (2003) who reported 3.94, 5.10,
3.38 and 4.0 billion sperm per ml in Awassi , Emirate Local and Emirate
Local x Chios, Sudan Desert sheep and Awassi rams respectively. Lower
values were recorded by Chiboka (1980) with West African Dwarf rams
and Loubser and Van Niekerk (1983) with Angora rams. The present
investigation revealed highly significant effect (P< 0.01) of summer
89
season on sperm cell concentration. This is in accordance with the
findings of many authors (Vijil, 1987; Daader, 1987; Fernandez, 1993;
Brewer et al., 1995; Alsayed, 2001; Gündogan and Demirci, 2003;
Gündogan, 2006) who reported significant dependency of sperm cell
concentration on the seasonal influences and not on the semen volume.
This indicated that the effect of season on testicular function is greater
than that exerted on accessory gland function (Salah et al. 1992). In the
present results, it can be considered that, the semen concentration in both
breeds fell within the range of good semen quality even in the hot
summer (Evans and Maxwell, 1987).
4-1-6 Live sperm count:
The mean values of live sperm cell percentage in this study
were 76.90±1.25 and 84.73±1.93 percent for Hamari and Kabashi
rams respectively. They were in line with those reported by Galil and
Galil (1982) in Sudan Desert sheep and Daader et al. (1987) in
Rahmani x Finnish Landrace rams. Higher values were recorded by
Dabas et al. (1997) in Patan-wadi rams, Manahil (1999) and Alsayed
(2001) in Sudan Desert sheep. Lower values were reported in
different breeds (Chiboka, 1980, in West African Dwarf rams;
Loubser and Van Niekerk, 1983, in Angora rams). There was a
highly seasonal effect (summer) on live sperm cells percentage in
90
rams of the two breeds under the present study. This is in agreement
with many authors (Chiboka, 1980; Vijil, 1987; Daader et al., 1987;
Fernandez, 1993; Dabas et al., 1997 and Alsayed, 2001). The
interaction term between breeds and seasons was significant. This
indicates that the ranking of the two breeds varied among seasons.
The percentage of the live spermatozoa in the present study (over
70%) was within the acceptable level needed for good fertility
(Gomes, 1977) even in the hot season. The increase in the percentage
of dead spermatozoa during summer agreed with the previous reports
with Sudan Desert sheep (Galil and Galil, 1982; Alsayed, 2001).
4-1-7 Sperm cells abnormalities:
The mean values of sperm cell abnormalities in this experiment
were 4.26±1.01 and 4.47±1.18 percent for Hamari and Kabashi rams
respectively. These values were in good agreement with the findings
reported by Dabas et al. (1997) in Patan-wadi rams and Manahil (1999)
in Sudan Desert sheep. Higher values were reported by many
investigators (Chiboka, 1980, in West African Dwarf rams; Loubser and
Van Niekerk, 1983, in Angora rams; Daader et al., 1987, in Rahmani x
Finnish Landrace rams; Kalous and Samkova, 1991, in Sumava rams).
Though, lower value was recorded by Demirci (1993) in Awassi rams.
The analysis of data revealed significant differences (P< 0.05) in the total
91
percentage of abnormal spermatozoa in the different seasons. This is in
agreement with many authors (Daader et al., 1987; Vijil, 1987,
Fernandez, 1993 and Gündogan, 2006). The incidence of head and tail
abnormalities were in accordance with the results recorded by Demirci
(1993) in Awassi rams. The incidence of mid-piece abnormalities in the
present study was in favour of those reported by Alsayed (2001) in Sudan
Desert sheep. The present study indicated that the incidence of loose
heads in both breed-types were higher than those of Alsayed (2001) in
Sudan Desert sheep. Statistical analysis showed a significant summer
effect (P< 0.05) on mid-piece, tail, acrosome abnormalities and highly
significant (p<0.01) on head abnormalities and incidence of loose heads
in both breeds. The best results were recorded during autumn and winter
compared to summer. However, the high ambient temperature during
summer season could be a direct cause in the elevation of sperm
abnormalities where temporary epididymal or accessory dysfunction
could occur (Fayemi and Adegbite, 1982). However, all the rams under
investigation had average spermatozoal abnormalities within the
recommended limits of good quality semen, as determined by Evans and
Maxwell (1987), even during the hot summer.
92
4-2 Scrotal circumference (Sc):
The mean values for Sc in the present study were
33.12±0.21 and 31.38±0.31 cm for Hamari and Kabashi rams
respectively. These values were similar to those obtained by Master
(1988) in Dohne Merino rams (32.2 cm), Demirci (1993) in Awassi
rams (30.75cm), Zhang et al. (1995) in Xinjiang Fine Wool rams
(33.34 cm) and Kafi et al.(2004) in Karakul rams (32.6). The
measurements of Sc of Hamari and Kabashi rams used in the present
experiment fell within the normal size of breeding rams (Master,
1988). In this study Sc was medium correlated with sperm
concentration (0.54) in Hamari rams and highly correlated with body
weight (0.70) in Kabashi rams. Similarly Trejo et al. (1990) have
reported 0.76 correlation coefficient for Sc with body weight in
Suffolk, Romney, Rambouillet, Pelibuey and Criollo rams. Weak
correlations of Sc with semen volume in the present study of 0.18 and
0.38 for Hamari and Kabashi rams respectively. These values were
not comparable to the values reported in the literature of 0.63 for
Manchega rams (Vijil, 1983); 0.98 for Awassi rams (Demirci, 1993)
and 0.84 for Xinjiang Fine wool rams (Zhang et al., 1995) for
correlations of Sc with semen volume. According to Mickelsen et al.
(1981), Downey et al. (1984), Dyrmundsson et al. (1989), Sackmann
93
and Schone (1990), Nwoha (1996), Gastel et al., (1995), Gündogan
and Demirci (2003); Melpomeni et al.(2004) and Gündogan (2006)
who reported significant seasonal variations on the testes volume . In
this study no significant difference was found between Sc
measurements in the two breeds in different seasons of the year. The
explanation for that may be that the rams in our study were physically
mature and they were effectively monitored not to fluctuate in their
body weight during the study and were kept indoors all year round
taking the same feeding levels and/or due to the in-significant
extreme temperature fluctuation and day light differences in the
experimental area. This is in agreement with Cameron et al. (1988),
Fernandez (1993), Gastel et al. (1995) and Bielli et al. (1997) who
suggested that the differences existed in seasonal variations in
testicular dimensions in adult rams due to the differences in feeding
levels. The interaction term between breeds and seasons was
significant. This indicates that the ranking of the two breeds varied
among seasons.
4-3 Reaction time (RT):
The mean values for the reaction time to the first mount in the
present study were 16.41±0.46 and 20.10±1.10 seconds for Hamari and
Kabashi rams respectively. They were lower than the mean values
94
reported by Zuzanna (1984) with rams (70.9 seconds, ranging from 30 to
166.7 seconds), Ibrahim (1997) with Emirates Local and Local x Chios
breeds raised under sub-tropical conditions (43.7 seconds) and Lezama
(2003) with Katahdin yearling rams (73 seconds). Regarding RT, the
breed had no significant effect but the winter exerted a highly significant
effect (P< 0.01). This is conforming to the findings of Trejo et al. (1990)
with Suffolk, Romney, Rambouillet, Pelibuey and Criollo rams raised on
the high plateau in Mexico. The shortest RT recorded was in summer and
autumn (14 seconds) in both breed-types, whereas, the longest time
recorded was in winter (45 seconds for Hamari rams and 35 seconds for
Kabashi rams). These values signify that the high ambient temperature of
summer did not completely depress the sexual activity of the tested rams.
This is in agreement with the results reported by Lindsay (1969) with
Merino rams, Ibrahim (1997) with Local Emirates and Local x Chios
rams and Gündogan and Demirci (2003) in two Turkish breeds. However,
there was a seasonal effect on RT but not to the extent of complete
depression as in temperate breeds and this implies that there was no
photoperiodic effect on this trait.
4-4 Freezability of spermatozoa:
Semen is usually evaluated both before and after freezing with the
aim of identifying sires and/or ejaculates with satisfactory semen quality.
95
Semen ejaculates are assessed in terms of sperm concentration, sperm
motility and morphology, whereas motility post-thawing is the only
parameter used to evaluate freezing procedures (Rodriguez and Larsson,
1998). The average percentage of post-thawing sperm motility in the
present study was 56.75±5.95 and 56.19±5.32 percent for Hamari and
Kabashi rams respectively. The results in this experiment showed that the
post-thawing motility levels were higher than those found by Loubser and
Van Niekerk (1983) with Angora rams (46.62%). However, our results
were lower to those found by other authors (Söderquist et al., 1997 and
Gil, 1999) in temperate breeds. The difference may be due to the
protocols and techniques used for semen dilution and freezing
(centrifugation). Similarly non-significant effect of breed and season
were found between Hamari and Kabashi breeds in post-thawing motility
(Loubser and Van Niekerk, 1983). Though, the results obtained in the
present study satisfy the criterion described for suitable frozen semen
needed for cervical insemination in both breed (Evans and Maxwell,
1987; Berg, 1999).
4-5 Rejection rate:
The incidence of rejected samples of semen before and after
freezing in the present study were 30.77, 32.44 and 15.38, 6.35 percent
for Hamari and Kabashi rams respectively. The data showed a significant
96
difference (P< 0.05) between the two breeds and between seasons of the
year in the rate of rejected semen samples after freezing, with better
results for Kabashi breeds (15.38 vs. 6.35). This could mean that Kabashi
rams spermatozoa were more resistant to low temperature during freezing
than that of Hamari rams. Similar results reported by Vlachos and
Tsakalof (1965) that the semen of Friesian rams was more sensitive to
low temperature during freezing than that of Chios. The significant
influence of winter season on semen freezability is in agreement with
finding of D´Alessandro and Martemucci (2003) with Leccese rams and
this is may be due to the seasonal variations in quantity and quality of
seminal plasma proteins (SP), and their differential protective effect
against cold-shock (La Falci et al.,2002; Dominguez et al.,2008). Autumn
and winter seasons were the most favourable periods to harvest high
quality semen for freezing and AI purposes.
4.6 Fertility following AI:
The pregnancy rates following oestrous synchronization and AI
using frozen-thawed semen achieved in the present study were 35.3% and
41.3% for Hamari and Kabashi rams. The results were comparable to
those reported by other authors (Dyrmundsson et al., 1989; Molinia et al.,
1996; Cappai et al., 1998; Naqvi et al., 1998; Sördequist, 1999; Sánchez-
Partida et al., 1999; Anel et al., 2003; Fair et al., 2005). Conventional
97
cervical insemination in ewes with frozen-thawed semen has not been
applied fully on sheep industry due to disappointing results (fertility 10 –
30%) (Windsor et al., 1994, Salamon and Maxwell, 1995, Maxwell and
Watson, 1996, Monika, 2006). Ewe breed has been shown to have a
major effect on pregnancy rate following cervical AI using frozen-thawed
semen. Conception rate values of 8, 28, 44, and 77% were reported for
Suffolk, Texel, Belclare and Finnish landrace breeds respectively
(Donovan et al., 1999). In Norway higher fertility result in Dala breed
61.4 and Spæl breed 71% were reported (Berg, 1999). The possible
reason for this difference is strongly dependant on breed and age of the
ewes and this is attributable to the morphometric parameters, such as the
external diameter and number of cervical folds, type of cervical orifice
and mucus secretion or depth of penetration of the cervix AI (Fukui and
Roborts, 1978; Berg, 1999; Donovan et al.2004). However, the lower
fertilizing rate following cervical AI must therefore be at least partially
due to impeded sperm transport through the genital tract (Salamon and
Maxwell, 1995). Although, laparoscopic AI is the only method that
ensures intrauterine application of frozen-thawed semen with satisfactory
fertility in sheep under field conditions (Salamon and Maxwell, 2000;
Fukui et al., 2007), laparoscopic AI has problems related to complexity,
high equipment costs and the need of trained technicians (Naqvi et al.,
1997; Anel et al.,2006). The higher fertility results that were obtained
98
after cervical insemination with fresh semen compared to frozen-thawed
semen in Sudan Desert sheep (Alsayed, 1996, 2001; El Mubark,2001;
Makawi and Manahil,2007), could be due to the reduced viability of
frozen spermatozoa resulting in low numbers of viable or undamaged
spermatozoa reaching the fertilization site (Monika, 2006).
4-7 Conclusions and Recommendations:
(1) Measurement of scrotal circumference is the useful test for
identifying rams that are suitable for breeding and artificial
insemination purposes.
(2) The data obtained clearly showed that the two breeds are
continuous breeding animals as they were capable of
producing semen of good quality all the year round.
However, seasonal fluctuation in semen characteristics were
observed but was not sharp as in temperate breeds.
(3) The semen collected from the two breeds was suitable for
freezing and recovery rate after thawing was within the
acceptable limits needed for cervical insemination.
(4) Semen of best quality was obtained during autumn and
winter which are the suitable periods to harvest semen for
freezing and AI purposes.
99
(5) The results obtained provided a new approach to the
cryopreservation of ram semen, and could positively
contribute to intensive sheep production and gene
conservation.
(6) Although conception rate achieved following cervical
insemination using frozen-thawed semen is relatively
low, but still it is an important objective of establishing
a cost-effective and widely applicable AI procedure for
acceptable pregnancy rates in sheep.
100
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Appendix (1): Climatic conditions in Khartoum during the experimental period.
Temperature (C˚) Month
Maximum Minimum Average
Humidity
(%)
Rainfall
mm
April 99 40.8 25.0 32.9 15 0.0
May 42.8 27.8 35.3 21 0.0
June 42.4 27.5 35.0 23 0.0
July 39.7 25.8 32.8 37 1.4
August 39.5 25.8 32.7 43 8.5
September 39.5 25.4 32.5 42 15.3
October 37.9 25.8 30.9 30 34.8
November 35.6 20.6 28.1 23 0.0
December 31.1 16.2 23.7 30 0.0
January2000 30.2 14.7 22.5 27 0.0
February 32.8 16.2 24.5 19 0.0
March 37.6 21.1 29.3 15 0.0
142
Appendix (2) Seminal characteristic Anova table
Source Variables df Mean square F Sig.
Season Semen volume
Mass activity
Individual motility
Sperm count
Live cells
2
2
2
2
2
.203
42.115
3091.889
12.064
1810.025
.875
47.719
30.654
23.075
29.223
.006
.250
.176
.139
.169
Breed Semen volume
Mass activity
Individual motility
Sperm count
Live cells
1
1
1
1
1
.344
.000
2.150
.746
646.478
1.481
.000
.021
1.427
10.437
.005
.000
.000
.005
.035
Season*breed Semen volume
Mass activity
Individual motility
Sperm count
Live cells
2
2
2
2
2
.100
1.701
10.690
.471
400.158
.432
3.367
.106
.900
6.461
.003
.023
.001
.006
.043
Error Semen volume
Mass activity
Individual motility
Sperm count
Live cells
287
287
287
287
287
.232
.505
100.864
.523
17776.481
143
Appendix (3) Sperm cell abnormalities Anova table
Source Variables df Mean square F Sig.
Season Abnormal mid-piece
Abnormal tails
Abnormal heads
Abnormal acrosome
Loose heads
2
2
2
2
2
1.551
8.397
29.188
1.264
37.154
3.138
4.358
13.937
1.919
21.652
.050
.017
.000
.156
.000
Breed Abnormal mid-piece
Abnormal tails
Abnormal heads
Abnormal acrosome
Loose heads
1
1
1
1
1
2.192
3.963
1.013
.113
6.544
4.435
2.057
.484
.172
3.814
.039
.157
.489
.680
.055
Season*breed Abnormal mid-piece
Abnormal tails
Abnormal heads
Abnormal acrosome
Loose heads
2
2
2
2
2
.076
2.227
4.019
.534
3.958
.154
1.156
1.919
.810
2.307
.857
.322
.155
.449
.108
Error Abnormal mid-piece
Abnormal tails
Abnormal heads
Abnormal acrosome
Loose heads
61
61
61
61
61
.494
1.927
2.094
.659
1.716