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Title: The anatomy of sheep cervix and the development of trans-cervical
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artificial insemination with frozen-thawed semen in ewe
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Short version of the title: Development of TCAI in sheep
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Author: Sukanya Leethongdee*
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Faculty of Veterinary and Animal Sciences, Mahasarakham University
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Tambon Talaad, Amphur Muang, Mahasarakham 44000 Thailand
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Phone/ Fax 043 742823 e-mail address: sukanya.l@msu.ac.th
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*corresponding author
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Abstract:
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The artificial insemination has an important role in the sheep industry and the
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sheep genetic improvement. The complexity of sheep cervix limits the
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development of the trans-cervical artificial insemination. The sheep cervix is a
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long convoluted tubular organ with the internal rings. The internal cervical
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rings form the funnel shaped-like of the cervical canal which is the
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physiological barrier situated inside the cervical canal. The cervical opening is
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formed by the internal cervical fold which varied to 5 types of cervical
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opening including duckbills, slit, rose, papilla and flap. The internal ring is
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arranged to grad 1, 2 and 3 according to their complexity of the internal fold
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alignment. These complicated structures of the anatomy of sheep cervix
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reduce the ability of the passage of the insemination pipette into the uterine
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body. The study of the cervical relaxation mechanism and the goat cervix
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anatomy is important for the development of trans-cervical artificial
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insemination in sheep. The administration of exogenous substance induces the
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cervical relaxation and enhances the possibility of the success of TCAI in
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sheep.
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Keyword: Trans-cervical artificial insemination, Frozen-thawed semen,
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cervix, cervical relaxation, ewe
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1. Introduction
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Artificial insemination (AI) of sheep is an advantageous management practice
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aimed at the genetic improvement at farm level and for programme of genetic
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selection. Furthermore AI has the potential for a significant impact on the
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sheep breeding industry. The main role of AI in sheep production is to
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increase the rate of genetic improvement and AI also contributes to achieve
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other goals e.g. allowing extensive use of the best available rams, therefore
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increasing selection pressure and the rate of response to selection. With AI,
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superior rams can be identified more easily through progeny testing. Because
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progeny testing involves large numbers of animals over long periods of time,
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sires under test may be too old or even dead by the time their progeny has
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proven valuable. Therefore, AI by speeding up the identification of superior
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rams at a younger age, results in faster genetic progress. The use of frozen
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semen for AI can also increase the rate of genetic progress by storing semen
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collected from young rams before their superiority is confirmed by progeny
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testing, therefore allowing the use of genetically superior semen more widely.
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AI ensures good paternal control and fertilization of groups of female by
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males of different genotypes is easily achieved. In addition AI takes advantage
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of oestrous synchronization with its precise control of ovulation and
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parturition and furthermore allows the advantage of out of season breeding.
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In addition to the value of AI with frozen semen for genetic improvement, AI
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is associated with other animal health benefits. This technique helps avoid
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disease transmission and allows the transport of semen, when the risk of
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disease prevents ram movement and AI reduces the risk of spreading sexually
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transmitted genital infections associated with natural mating. There are some
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dangers with the widespread use of AI when used extensively with a limited
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number of sires mainly from a reduced genetic variation in the population.
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Moreover, it is possible that hereditary defects and undesirable traits can be
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rapidly disseminated.
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2. Artificial insemination techniques
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There are three AI techniques that have been used in sheep industry. These are
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deep vaginal insemination, the laparoscopic intrauterine insemination and
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cervical insemination. A fourth method for sheep, Tran-cervical artificial
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insemination (TCAI), is not widely used. The methods differ in their
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complexity and success rate. The fertility rates following the vaginal, cervical
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and laparoscopic insemination all vary with the insemination technique used
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as well as with farm, age, male, number of insemination per ewe, lambing-
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insemination interval, technician, flock and management conditions (Paulenz
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et al., 2005; Anel et al., 2005).
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2.1 Vaginal insemination
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This method involves depositing semen deep in the vagina without any
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attempt to locate the cervix.
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Vaginal insemination using fresh diluted semen is the simplest and quickest
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method but requires a large semen dose (150-400 million spermatozoa per
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insemination) (Figure 4a). Vaginal insemination using fresh semen gives an
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acceptable lambing rate. Unfortunately the transportation and preservation of
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fresh semen limits its use among sheep farmers. Therefore AI, using F-T
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semen is an alternative and accepted option. Vaginal insemination using F-T
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semen gives variable lambing rates; 17% (Tervit et al., 1984), 17.6%
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(Maxwell and Hewitt, 1986), 31.25 % (Anel et al., 2005) and 67.4% (studied
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in 543 Norwegian crossbred ewes, inseminated with 200 million spermatozoa)
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(Paulenz et al., 2005).
Semen is deposited in the anterior vagina.
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Cervical insemination with F-T semen also gives low fertility. Cervical
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insemination using fresh semen gives a higher pregnancy rate than cervical
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insemination using F-T semen (Donovan et al., 2004); 76% compared with
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46% (Irish breed) and 36% (Norwegian breed). The use of F-T semen and
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cervical insemination gave better fertility than vaginal insemination with F-T
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semen. The reports illustrating low fertility rates following vaginal
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insemination with F-T semen such as Maxwell and Hewitt (1986) who
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reported a 18.4% pregnancy rate after cervical insemination with F-T semen
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compared to 17.6% after vaginal insemination with F-T semen (100 million
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spermatozoa per insemination). Paulenz et al. (2005) reported 71% pregnant
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based on day 25 non-return to oestrus and a 67.4% lambing rate following
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vaginal insemination with F-T semen that was significantly different from a
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75.4% non return rate and a 72.7% lambing rate following vaginal
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insemination with F-T semen (200 million spermatozoa per insemination).
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The site of insemination influences fertility and cervical insemination has a
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higher fertility than vaginal insemination when using the F-T semen.
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2.2 Intra-cervical insemination
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Intra-cervical insemination using fresh diluted semen is commonly used in AI
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of sheep. When performed properly, cervical insemination with fresh diluted
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or undiluted semen results in high fertility, whereas the fertility obtained
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following intra-cervical insemination with frozen-thawed (F-T) semen is poor
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(Figure 4b). Intra- cervical insemination is performed by insemination at the
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cervical opening or at the deepest possible intra-cervical site that is easily
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accessible without attempting to force the inseminating pipette into the
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cervical canal (Ayad et al., 2004; King et al., 2004). In 31% of ewes the
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inseminating pipette passes up to 1 cm. into the cervical canal, up to 2 cm in
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another
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inseminating pipette pass more than 3 cm beyond the cervical opening
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(Eppleston and Maxwell, 1995). The depth of penetration is related to breed
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(Kaabi et al., 2006) and age of ewe (Eppleston and Maxwell, 1993; Kaabi et
31% and up to 3 cm in 30%.
In only 8% of ewes did the
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al., 2006; Eppleston and Maxwell, 1995). In older ewes, the cervix is longer
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and wider with looser folds, allowing easier passage of an inseminating
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pipette. The depth of insemination has an effect on fertility and pregnancy rate
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and lambing rates increase as the depth of insemination into the cervix
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increases (Salamon and Maxwell, 1995; Halbert et al., 1990; Eppleston and
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Maxwell, 1995). The pregnancy rate was 11.7% when F-T semen was
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deposited 0-1 cm into the cervix. However as the depth of insemination
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increased the pregnancy rate increased, 13.7% when F-T semen was deposited
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at 1-2 cm, 22.2 % when F-T semen was deposited at 2-3 cm, and 34.8% when
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F-T semen was deposited beyond 3 cm in the cervix. The corresponding
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lambing rates were 15.5%, 24.1%, 24.2% and 75% respectively (Eppleston
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and Maxwell, 1993).
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2.3 Laparoscopic intrauterine artificial insemination
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Semen is deposited directly into the uterus through the uterine wall with the
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aid of a laparoscope. Sedation and local anesthesia are required. Fertility and
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pregnancy rates are high with either fresh or F-T semen. A lower number of
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spermatozoa can be used, typically 40 to 80 million spermatozoa per
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insemination (Figure 4c). The complex anatomy of the cervix limits the
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passage of an inseminating pipette into the cervical canal and causes difficulty
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with transport of spermatozoa through the cervix. The difficulty of cervical
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passage can be overcome by direct uterine insemination using laparoscopy
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(Killeen and Moore, 1970). Fertilization rates 72 h after laparoscopic
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insemination (92.5%) with fresh semen are greater than after cervical/trans-
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cervical insemination (28%) (Sayre and Lewis, 1997). Therefore this
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technique allows the effective use of F-T semen. The fertility of F-T
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spermatozoa is higher after laparoscopic insemination than after cervical or
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trans-cervical insemination (Sanchez-Partida et al., 1999). Laparoscopic
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insemination using F-T semen has contributed to improved genetic selection in
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sheep breeding. However it has disadvantages, the main disadvantages are the
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high cost due to the technical expertise required, the equipment is expensive
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and easily damaged and it may become unacceptable on animal welfare
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grounds and legislation. A low cost and less invasive technique that gives
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acceptable fertility using F-T semen is required.
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Even though there has been a lot of research attempting to improve these AI
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results following cervical insemination there are only two general commercial
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categories that have been used in sheep AI 1) using refrigerated semen (15˚C)
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with superficial intra-cervical insemination and 2) using F-T semen with
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laparoscopic insemination (Anel et al., 2006). Unfortunately the laparoscopic
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insemination is costly and not welfare friendly.
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desirable to develop intrauterine trans-cervical AI which allows the use of F-T
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semen to be inseminated into the uterus via the vagina and cervix.
Therefore it is highly
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2.4 Trans-cervical artificial insemination (TCAI)
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Trans-cervical artificial insemination (TCAI) is a method of insemination
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where semen is deposited deep in the cervix or even into the uterus via the
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cervix (Figure 5). This method involves depositing semen as deeply as
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possible in the cervix. The greater the depth of insemination, the higher the
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expected pregnancy and lambing rates (Eppleston and Maxwell, 1993;
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Salamon and Maxwell, 1995). A non-return rate of 58% following deep
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cervical insemination with F-T semen has been reported (Donovan et al.,
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2004). It is likely that the anatomical complexity of the sheep cervix limits the
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success of TCAI. Histological examination showed damage to the epithelial
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lining of the cervical canal following cervical penetration with an unmodified,
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conventional straight inseminating pipette, either into the uterus or the middle
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of the cervix.
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There are varying degrees of damage to the cervical lining over the length of
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the cervix canal (Campbell et al., 1996). A TCAI catheter has been developed
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that is said to prevent cervical trauma during trans-cervical passage (Wulster-
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Radcliffe and Lewis, 2002). The difficulty of traversing the cervix severely
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limits the use of TCAI because it causes cervical trauma and impairment of
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transport of spermatozoa. When the new TCAI catheter was used in a
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comparison with laparoscopic AI, using the F-T semen the results showed that
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there was no difference between the techniques for ovum and embryo
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recovery rates (mean = 87.3%), fertilization rates (59.3%) or Day 3 pregnancy
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rates (mean = 66.6%). This suggests that in the future TCAI may be able to
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replace laparoscopic AI in sheep. However the fertility rate after TCAI with F-
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T semen is lower - compared to laparoscopic insemination, although both
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techniques deposit semen in the uterus. Wulster-Radcliffe et al. (2004)
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reported that TCAI with F-T semen had a much lower fertility rate than
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laparoscopic AI; the pregnancy rate at day 30 was 5% versus 46% and at day
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50 it was 4% versus 41%, suggesting there are other factors that influence
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fertility after TCAI such as ewe breed (Donovan et al., 2004), stress from
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animal restraint or season (Langford et al., 1983). The study of alternative
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methods that overcome the poor fertility following TCAI with F-T semen is
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continuing. The use of the exogenous cervical dilatators in sheep such as
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oxytocin or oestradiol (Khalifa et al., 1992; Stellflug et al., 2001) have been
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investigated but a much better understanding of sheep cervical physiology and
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the mechanism of natural cervical dilatation at oestrus is required to facilitate
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the aim of developing and effective method of TCAI for sheep with F-T
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semen.
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3. The anatomy of the sheep cervix limits the TCAI
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The cervix is the most caudal portion of the uterus and its constricted lumen is
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surrounded by a thick musculo-connective tissue wall (Moré, 1984). The
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average length of the sheep cervix is variable depending on the age and breed
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of ewe (Kaabi et al., 2006). The average length of the cervical canal studied in
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the Canadian crossbreed ewe is 6.7 ± 1.1 cm and contains 4.9 ± 1.0 funnel-
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shaped rings (Halbert et al., 1990). The average cervical length studied in
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Merino, Castellana, Assaf and Chura is 6.86 cm and contains an average of
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4.16 cervical rings (Kaabi et al., 2006). The average cervical length of the
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Indian native breeds (Malpura and Kheri) in ewe lambs and adult ewes are 3.8
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± 0.12 cm and 5.3 ± 0.15 cm, respectively. The average number of rings in the
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cervices of ewe lambs and adult ewes are 3.2 ± 0.19 and 3.4 ± 0.22 (Naqvi et
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al., 2005). This agrees with the report by Kaabi et al. (2006), that in younger
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ewes the cervix is shorter and narrower, but Kaabi et al. (2006) noticed that
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younger ewes have more cervical rings than older ewes, suggesting the
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morphology of the cervix depends on the age of the ewe. The average cervical
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length of the Chura breed is less, the cervix narrower and has more rings than
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the Merino and Castellana breeds (Kaabi et al., 2006). In the older multiparous
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ewe, the cervix tended to become longer and wider and with loose rings.
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In the ewe, the cervical lumen has a convoluted and tortuous structure that
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looks like a corkscrew consisting of internal cervical rings (Halbert et al.,
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1990; Kaabi et al., 2006; Naqvi et al., 2005) (Figure 1). The internal folds of
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the cervix form a funnel-like shape with the narrow opening projecting
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caudally into the cervical lumen (Naqvi et al., 2005; Halbert et al., 1990). It is
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common that the second fold is eccentric to the other concentric folds and thus
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acts as an anatomical and physiological barrier (Moré, 1984). The cervical
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lumen is misaligned in 74.43% of ewes with the presence of eccentric folds.
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The most common eccentricity occurs at the second cervical ring (75%), but
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the third (14%) and first ring (11%) can also be eccentric (Kaabi et al., 2006).
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The alignment of cervical rings can be graded by their degree of completeness
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and inter-digitation (Kershaw et al., 2005) (Figure 2):
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Grade 1 the cervix has a series of complete aligned cervical rings lying across
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the lumen with no inter-digitation of the cervical rings.
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Grade 2 the cervix has a mixture of complete folds and incomplete cervical
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rings that lay partially across the lumen and inter-digitate with one another,
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obscuring
the central lumen.
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Grade 3 the cervix has predominantly incomplete and inter-digitating cervical
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rings that are not aligned.
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The average external diameter of the cervix is 1.36 cm. (Kaabi et al., 2006).
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The diameter of the cervical lumen is between 1.8 to 6.0 mm. The narrowest
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point is commonly found at the second, third or fourth rings. The cervix opens
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caudally into the vagina at the external os of the cervix. Sheep show high
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variation in the anatomy of the cervical opening. The first and second folds of
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the cervical rings form the shape of the cervical opening which varies with age
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and probably parity. The ovine external os cervix has been classified on the
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basis of its morphology (Kershaw et al., 2005; Halbert et al., 1990) (Figure 3):
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Duckbill: There are two opposing cervical folds protruding into the vagina to
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form a slit like os.
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Flap: There is one cervical fold protruding into the vagina that form a flap that
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lies over the cervical opening, thus causing difficulty when attempting to
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locate the cervical os.
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Rosette: There are several cervical folds that form a rosette of vaginal folds
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around the cervical opening.
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Slit: There are no cervical folds protruding into the vagina but there is a slit-
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like
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Papilla: There is a single cervical fold protruding into the vagina with the
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external os as its apex.
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Spiral: The cervical folds form a spiral which protrudes into the vagina.
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The papilla form is more frequent in younger ewe (< 2 year old), and the flap
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like form in older ewes (> 3 year old) (Kaabi et al., 2006) which may be the
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consequence of lambing (Dun, 1995). The rosette type of cervical opening is
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found commonly in adult ewes and the papilla type of cervical opening is
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commonly found in younger ewes (Kershaw et al., 2005). The anatomy of the
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cervical os type does not change with the stage of the oestrous cycle and its
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appearance is probably determined by genetic factors and by the mechanical
opening to the cervix on the anterior wall of the vagina.
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consequences of parturition (Kershaw et al., 2005; Kaabi et al., 2006).
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Because the second fold is consistently out of alignment with the first fold it
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effectively closes the cervical canal at that point and makes it quite difficult
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and sometimes impossible to introduce an inseminating pipette into the cervix.
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Cervical penetrability is positively correlated with the diameter and the
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cervical lumen diameter and cervical penetrability is negatively correlated
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with the number of cervical rings (Kaabi et al., 2006). The complexities of
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sheep cervical anatomy limit the passage of an inseminating pipette. However,
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cervical penetrability varies during the oestrous cycle (Kershaw et al., 2005)
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suggesting that the physical characteristics of the cervix and cervical dilatation
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are hormone-dependent. The study of the mechanism of cervical dilatation
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during the peri-ovulatory period is required if we are to develop a practical
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method for TCAI.
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4. The application of exogenous substances to relax the cervix
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Physiological cervical ripening is characterised by a diffuse loosening of the
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collagenous connective tissue with widely scattered collagen fibrils and an
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increased amount of extracellular ground substance (Rath et al., 1993; Rath et
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al., 1990). Therefore cervical dilatation requires a change in collagen within
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the cervical stroma (fibroblasts and smooth muscle) from the highly organized
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network of tightly bound collagen fibrils under the influence of high
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progesterone levels to a much looser arrangement at oestrus (Calder, 1994)
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that may facilitate cervical passage of an AI pipette. Therefore there have been
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numerous attempts using exogenous substances, to dilate the cervix at the
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oestrus and facilitate trans-cervical AI.
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The cervix naturally dilates slightly at oestrus, at a time when progesterone is
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low and oestradiol and oxytocin are high and effecting uterine contractibility.
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Cervical dilatation accompanies uterine contractility during labor, at a time
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when progesterone is declining as well as oestrogen and oxytocin rising
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(Challis et al., 1983). The use of exogenous oxytocin to increase cervical
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dilatation at oestrus was investigated (Khalifa et al., 1992). The injection 200,
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400 or 600 IU of oxytocin at 44h and 52h after the removal of a progestagen
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pessary facilitated passage of a stainless steel rod into the uterus. In addition
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the combination of oxytocin with 100 or 200 µg of oestradiol-17β (E2)
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injected 9 days after the removal of a progestagen pessary also allowed
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passage of a stainless steel rod into the uterus (Khalifa et al., 1992), suggesting
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that increased secretion of oxytocin and E2 at oestrus reduced the difficulty of
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passing a pipette through the cervix. However the mechanism of oxytocin-
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induced cervical dilatation is not known.
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Another report revealed that a combination of oxytocin and E2 facilitated
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trans-cervical embryo transfer in the ewe. The ewes received 100 µg of E2 by
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the intravenous injection 7d after the onset of oestrus and then 12h later, the
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ewes received 400 IU of oxytocin (Wulster-Radcliffe et al., 1999). This
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combination of oxytocin and E2 induced the relaxation of the cervix resulting
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in the increase of the success of the embryo transfer via the trans-cervical
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route (Wulster-Radcliffe et al., 1999). The application of oxytocin is likely to
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facilitate cervical dilatation but oxytocin may affect reproductive performance.
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This report also showed that the combination of oxytocin and E2 did not affect
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luteal function (Wulster-Radcliffe et al., 1999). The effect of exogenous
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oxytocin on the cervical dilatation and its effects on reproductive variables
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were investigated (Stellflug et al., 2001) and exogenous oxytocin tended to
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reduce the ovulatory interval. Cervical manipulation following oxytocin
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decreased fertilization rate, however cervical manipulation alone did not affect
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the fertilization and lambing rates. Furthermore, exogenous oxytocin
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decreased the pregnancy–specific protein B and lambing rates in ewes
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(Stellflug et al., 2001). Thus it seems that exogenous oxytocin is not a
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practical solution to the problem of trans-cervical AI. In another experiment
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that investigated the effect of exogenous oxytocin on the fertilization rate that
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followed laparoscopic or TCAI (Sayre and Lewis, 1997). Ewes were
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inseminated laparoscopically or trans-cervically with 200 million sperm 54 h
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after removal of progestagen pessaries. Thirty min before AI the ewes were
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injection with 200 IU of oxytocin. Fertilisation rates 72 h after AI were lower
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in ewes following TCAI compared to ewes inseminated by laparoscopic AI
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(Sayre and Lewis, 1997) indicating exogenous oxytocin did not but that TCAI
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per se did, reduce fertilization rates (Sayre and Lewis, 1997). The lambing rate
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(percentage of treated ewes lambing) and litter size (lambs per ewe lambing)
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following the intrauterine insemination with frozen semen (0.2 ml of 400
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million per ml) were tested with and without oxytocin (10 IU given by
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intramuscular injection) prior to fixed-time insemination (King et al., 2004).
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Oxytocin did not permit complete cervical penetration in any ewes and
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lambing rate but not the litter size, was reduced by oxytocin (King et al.,
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2004). It is likely that oxytocin is able to dilate the cervix allowing access to
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the uterus during conventional cervical insemination.
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6. Conclusion
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The TCAI is the artificial insemination technique which allows the potential of
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the using of frozen thawed semen in sheep. The frozen thawed semen is
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deposited directly intrauterine via the passage of insemination pipette through
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the cervical canal. Unfortunately, the anatomy of the cervix of sheep limits the
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development of the TCAI. The sheep cervix is a long tubular fibrous organ. It
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is composited by the layers of smooth muscle and the connective tissues. The
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sheep external os cervix has been classified on the basis of its morphology.
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The internal folds of the cervix form a funnel-like shape with the narrow
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opening projecting caudally into the cervical lumen which prevents the
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passage of the insemination pipette through the cervical canal. The application
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of the exogenous substance to relax the cervix provides the depth of the
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passage of the cervical penetration which leads to the possibility of the trans-
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cervical artificial insemination in ewe.
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Figure 1 The internal structure of the cervical canal. The silicones casts
represent the funnel-like shape of the internal cervical ring. (Naqvi et al.,
2005)
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Figure 2 The internal cervical rings (a) grade 1, (b) grade 2, and (c) grade 3
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The yellow arrows illustrate the direction of the insemination pipette (Kershaw
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et al., 2005).
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Figure 3 The variation of the sheep cervical opening (a) duckbill, (b) slit, (c)
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rose, (d) papilla and (e) flap (Kershaw et al., 2005).
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a) The vaginal artificial insemination: The
semen is deposited at the cervical opening. The
red arrow represents the insemination pipette
direction.
b) The cervical artificial insemination: The
semen is deposited into the cervical canal. The
red arrow represents the insemination pipette
direction.
c) The laparoscopic intrauterine artificial
insemination. The semen is deposited directly
intrauterine via the laparoscopic operation.
Figure 4 The artificial insemination technique in sheep production a) vaginal artificial
insemination b) cervical artificial insemination c) laparoscopic intrauterine artificial
insemination.
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Uterine horn
Uterine body
cervix
Fertilisation site
Figure 5 The trans-cervical artificial insemination. The semen is deposited
into the uterine body. The insemination pipette is passed into the uterine body
through the cervical canal. The red arrow represents the direction of the
insemination pipette.
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