Theriogenology 103 (2017) 30e35

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Theriogenology
journal homepage: www.theriojournal.com

Semen deposition by cervical, transcervical and intrauterine route for

fixed-time artificial insemination (FTAI) in the ewe
~ oz b, c, A. Mezzalira a, A. Menchaca b, *
R. Casali a, A. Pinczak b, F. Cuadro b, J.M. Guillen-Mun
a rio de Reproduça
Laborato ~o Animal Assis Roberto de Bem, Universidade do Estado de Santa Catarina - UDESC, Centro de Ci^ rias e CAV,
encias Agroveterina
Avenida Luiz de Camo ~es, 2090, Lages, Brazil
b
Fundacion IRAUy, Instituto de Reproduccion Animal Uruguay, Cruz del Sur, 2250, Montevideo, Uruguay
c
Universidad Autonoma Agraria Antonio Narro-Unidad Laguna, Torreo n, Coahuila, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Semen deposition through the cervix into the uterus is a difficult technique in ewes and represents the
Received 24 April 2017 main limiting factor for insemination in this species. The objective of this study was to evaluate the
Received in revised form pregnancy rate achieved with a new transcervical insemination method in comparison with conven-
2 July 2017
tional cervical and laparoscopic intrauterine techniques. A total of 586 multiparous Corriedale ewes were
Accepted 19 July 2017
Available online 26 July 2017
synchronized for fixed time artificial insemination (FTAI) performed by cervical, transcervical, or intra-
uterine route at 46e50 h or 52e56 h after progesterone device removal in a 3
2 factorial design.
Pregnancy rate was affected by the insemination technique and by the moment of FTAI (P < 0.05),
Keywords:
Sheep
without interaction (P¼ NS). Overall, the fertility was improved as semen deposition was deeper and
Intracervical insemination was delayed. For transcervical insemination, pregnancy rate was intermediate (42.3%; P¼
Sperm NS) between cervical and intrauterine route (36.0% and 50.2%; P < 0.05), and was greater for those ewes
Estrus inseminated beyond 4 cm into the cervix (60.0% versus 35.1% for insemination beyond or within 4 cm
Synchronization into the cervix, respectively; P < 0.05). Semen deposition beyond 4 cm into the cervix was achieved only
in 28.8% of the females receiving transcervical insemination. This method was more time-consuming
than cervical or laparoscopic insemination (11.4 ± 1.6 versus 85.5 ± 7.5 and 56.8 ± 5.6 ewes insemi-
nated per hour, respectively; P < 0.05). In summary, greater pregnancy rate using FTAI is obtained when
semen is placed into the uterus, which was achieved in all females only through laparoscopy. Further
improvements are required for transcervical insemination to be applied in large-scale FTAI programs in
Corriedale ewes.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction limiting factor for insemination is the difficulty to go through the

cervix with the insemination pipette for intrauterine semen
Artificial insemination is a high-impact technology in ruminant deposition. The cervix of the ewe is a small, narrow, rigid and
industry because it increases genetic improvement, makes easier tortuous structure that makes difficult cannulation and deposition
the international trade of males and the conservation of genetic of semen into the uterus [3,4]. For this reason, usually semen is
resources, allows a better control of reproduction, and contributes located in the entrance of the cervix trough the vagina canal (cer-
with animal health by controlling several infectious diseases. In vical insemination) or injected into the uterine lumen through the
addition, the possibility to perform fixed time artificial insemina- uterine wall by laparoscopy (laparoscopic insemination).
tion (FTAI; i.e. without estrous detection) further promotes the use The intrauterine insemination assisted by laparoscopy is an
of this tool by technicians and farmers making its implementation effective technique adapted for sheep in the 1980s [5]. This is the
much easier (see review in sheep and goats: Menchaca and preferred technique mainly when frozen semen is used, since
Rubianes [1]; cattle: Bo et al. [2]). In ewes, unlike cows, the main normally fertility of cryopreserved sperm by cervical insemination
is extremely poor [6]. In addition, intrauterine insemination in-
duces greater pregnancy rate than cervical insemination also with
fresh semen [7]. For this reason, intrauterine insemination at pre-
* Corresponding author. fixed time performed by laparoscopy is used in large-scale
E-mail address: [email protected] (A. Menchaca).

http://dx.doi.org/10.1016/j.theriogenology.2017.07.021
0093-691X/© 2017 Elsevier Inc. All rights reserved.
 R. Casali et al. / Theriogenology 103 (2017) 30e35 31

programs in several countries. However, although laparoscopy is a twice 7 d apart with the second dose administrated at time of DICO
minimally invasive procedure, it requires veterinary expertise, removal in the females. The females were randomly assigned to one
implies animal welfare concerns, and is more demanding in terms of three insemination techniques by cervical, transcervical, or in-
of equipment and labor than cervical insemination. Even though trauterine route by laparoscopy. The FTAI was performed for each
several alternative approaches have been proposed to replace the technique in the morning or in the afternoon of Day 8 (i.e. 46e50 h
use of laparoscopy, this technique continue being the default and 52e56 h after device removal) in a 3
2 factorial design. Fresh
method when obtaining greater pregnancy rate is mandatory. semen was collected by artificial vagina from four Awassi rams and
Transcervical insemination technique to perform intrauterine only those ejaculates within acceptable parameters regarding vol-
semen deposition through the cervix is not an usual practice in ume (0.75e2 ml), sperm concentration (>3
109 spermatozoa/ml),
sheep. In the 1970s, Fukui and Roberts [8] described a method and sperm motility (>70% motile cells) were used. The sperm
consisting of the fixation of the cervix with long forceps and a ball- concentration was determined with a spectrophotometer (SDM1,
tipped 17-gauge hypodermic needle attached to an inseminating Mintüb, Tiefenback, Germany) and after evaluation semen was
pipette. This was introduced through the external os into the cer- diluted in skim UHT milk and used in pools from all the rams (one
vical canal. Some years later, in the 1990s Halbert et al. [9] evalu- ejaculated per ram) to prevent individual effect of the rams on
ated four methods of restraint, four vaginal specula, three forceps fertility. Each pool of semen was divided in three aliquots to be used
and four instruments for transcervical passage, and developed a for cervical, transcervical or intrauterine FTAI, and the dilution rate
method that they named the Guelph System, which was later was adjusted to use 150
106 spermatozoa in 0.1 ml, 100
106
assessed in terms of lambing rate with variable success [10]. spermatozoa in 0.2 ml, or 75
106 spermatozoa in 0.2 ml per ewe,
Different approaches, techniques, devices and drugs have been respectively. The dilution rate was established according the sperm
proposed by several authors in diverse conditions to improve the concentrations usually reported for these techniques [21],
passage of the ovine cervix [11,12,13,14]. The fertility outcomes are regarding the well-known concept of as deeper the insemination
contradictory among reports, varying from 0% [15] to 70% [16] of site is, the sperm concentration required is lower. Diluted semen
pregnancy rate, and some of them are presented as proof of concept was maintained at 30  C and the ewes were inseminated within 1 h
that sometimes are not followed by validation or field trials with from semen collection (i.e. the three techniques simultaneously).
enough number of females, breeds and ages. Recently, promissory After this period, the remaining semen was discharged and
outcomes have been reported with a novel transcervical approach collected again for a new insemination session. Pregnancy rate was
for uterine flushing and embryo transfer in sheep and goats [17,18]. determined by transrectal ultrasonography using a 5.0 MHz
This technique consist in the traction and fixation of the cervix transducer (Aloka 500SSD, Tokyo, Japan) 30e31 days after
through the vagina and catheterization assisted by rectal or vaginal insemination.
manipulation using one or two fingers. Although this technique has
been reported for insemination in sheep, little information is 2.2. Insemination techniques and semen deposition
available with its use in comparison with conventional cervical or
laparoscopic insemination. In addition, information with this Cervical, transcervical and intrauterine laparoscopic insemina-
technique is even scarce associated with FTAI, and several factors tion was performed by three veterinarians with enough experience
such as time of insemination, dose of sperm required, and time using each technique in large-scale FTAI programs. The three
consuming during procedure should be defined. techniques were conducted at the same time in three insemination
The objective of this study was to evaluate pregnancy outcomes stations mounted for cervical, transcervical and intrauterine
obtained with insemination by transcervical technique compared insemination.
with conventional cervical insemination and intrauterine insemi- For cervical insemination (n ¼ 239), the females were restrained
nation by laparoscopy, evaluating the feasibility to apply this with the hindquarter upwards on the railing fence panel as per-
method in large-scale FTAI programs in sheep. formed routinely. The insemination was done using a vaginoscope
with light source incorporated and a multidose insemination gun
2. Material and methods (Walmur, Montevideo, Uruguay) that allow semen deposition at the
end of the vagina in the external os of the cervix.
2.1. Animals and treatments for FTAI For transcervical insemination (n ¼ 104; Fig. 1), the females
were placed with the hindquarters upward maintained on an easel,
The study was conducted during the breeding season in and with the aid of a duck-billed speculum and external light
Uruguay (May, 33 S) and all the procedures were approved by the source, the cervix was located to be sprayed with 2% lidocaine
Institutional Animal Care Committee of IRAUy certified by the Na- hydrochloride. After that, the cervix was fixed outside the specu-
tional Council of Animal Care of Uruguay. A total of 586 multiparous lum by using a non-traumatic Allis forceps, and then, clipped
Corriedale ewes with a body condition score of 3.0 ± 0.1 0.5e1 cm laterally to the external os with two 26 cm Pozzi forceps
(Mean ± SEM; scale 0e5) [19] were synchronized in two consec- [18]. Cervical traction was performed gently and the cervix was
utive days to receive FTAI after a short-term progesterone protocol positioned to enable complete manipulation directly with the fin-
described by Menchaca and Rubianes [1]. The treatment for FTAI gers. The cervix catheterization was performed with a thin metal
consisted of the insertion (Day 0) of an intravaginal silicone device cannula of 2 mm in diameter (Wago, SP, Brazil) designed for con-
(0.3 g progesterone, DICO®, Syntex, Bs As, Argentina), and one dose taining a 0.25 ml semen straw, which was threading by external
of eCG (300 IU, Novormon, Syntex) and prostaglandin F2alpha manipulation with two fingers (i.e. thumb and index). After semen
analog (125 mg sodium cloprostenol, Ciclase DL, Syntex) given im on discharge, the deep of penetration was measured in the cannula.
Day 6 in the morning at device removal. The DICO® devices had The transcervical technique is depicted in Fig. 1.
been previously validated for sheep with similar pharmacokinetics Intrauterine insemination (n ¼ 243) was performed by lapa-
and pharmacodynamics [20] and fertility [7] than CIDR-G® devices. roscopy (Karl Storz, Tuttlingen, Germany). All ewes were fasted and
The ewes were exposed to androgenized Corriedale wethers from had restricted access to water for 12 h before the procedure. For
device removal to insemination in a 10:1 females: male ratio. The insemination, the females were restrained in cradles with the rear
wethers were previously treated with testosterone (200 mg of legs lifted to an approximate 40e45 angle. The trocar and cannula
testosterone propionate, Dispert, Montevideo, Uruguay) given were inserted into the peritoneal cavity to the left and right of the
32 R. Casali et al. / Theriogenology 103 (2017) 30e35

Fig. 1. Transcervical insemination in the ewe. The female is placed with the hindquarters upward maintained on an easel, and with the aid of a duck-billed speculum and an
external light source the cervix is located to be sprayed with 2% lidocaine hydrochloride. The instruments are showed in the upper panel (A). The entrance of the cervix is fixed
cranial to the speculum by using a non-traumatic Allis forceps (B), to be clipped 0.5e1 cm laterally to the external os with two 26 cm Pozzi forceps for gently cervix traction and
exteriorization (C). The cervix is positioned to enable complete manipulation directly with two fingers (e.g. thumb and index) for catheterization with a thin metal cannula of 2 mm
diameter and blunt tip (D). After cannulation, 0.25 ml semen straw is discharged as deeper as possible into the cervix or directly into the uterus lumen (E).
 R. Casali et al. / Theriogenology 103 (2017) 30e35 33

Table 1
Pregnancy rate obtained with cervical, transcervical or intrauterine laparoscopic FTAI performed at 48 h (46e50 h) or 54 h (52e56 h) from progesterone intravaginal device
removal.

Pregnancy rate (pregnant/FTAI ewes) No. fetuses/pregnant ewes No. ewes FTAI per hour

FTAI FTAI P value Total

48 h 54 h FTAI

Cervical FTAI 31.1%a 42.3%a 0.07 36.0%a 1.20a 85.5 ± 7.5a

150
106 sperm cells (42/135) (44/104) (86/239) (103/86)
Transcervical FTAI 32.7% ab 53.1% ab <0.05 42.3% ab 1.27a 11.4 ± 1.6b
100
106 sperm cells (18/55) (26/49) (44/104) (56/44)
Intrauterine FTAI 44.1%b 56.9%b <0.05 50.2%b 1.21a 56.8 ± 5.6c
75
106 sperm cells (56/127) (66/116) (122/243) (148/122)

For the same column, different superscripts P < 0.05.

middle line, approximately 5 cm cranial to the udder. The perito- 4. Discussion

neal cavity was inflated with carbon dioxide to help visualize the
uterus with the aid of the laparoscope and separate it from the The current study demonstrates that greater pregnancy rate is
abdominal wall. Once the uterus was in the correct position, the obtained when intrauterine semen deposition is performed, which
operator punctures the uterine horn and the semen was injected was achieved mainly with laparoscopy. As an overall outcome,
directly into the lumen of the uterus using an insemination pipette insemination by laparoscopy allowed acceptable pregnancy rate
connected to a 1 ml syringe. The same procedure was repeated on with a lower sperm dose and with a reasonable time required per
the other uterine horn. ewe. The pregnancy rate with the transcervical route was inter-
mediate, demanding more time and animal handling than the other
2.3. Statistical analysis methods. Fixed time artificial insemination performed by cervical
route with double semen dose than laparoscopy, resulted in
Pregnancy rate and number of fetuses per pregnant ewe was significantly lower pregnancy rate.
compared by GLMM of the Infostat package [22] with the insemi- Although previous studies have showed that intrauterine
nation technique and the moment of FTAI (48 h or 54 h) included as insemination by laparoscopy results in greater pregnancy rate than
fixed effects with its interaction, and the animal as random effect. cervical insemination, also using fresh semen and FTAI [7], little
The significance level was defined for a P value < 0.05. information about these three methods associated to FTAI have
been reported (with no data available in Corriedale ewes). In
3. Results Australian Merino sheep, Windsor et al. [23] compared conven-
tional methods using laparoscopic and cervical insemination versus
Pregnancy rate was affected by the insemination technique and transcervical procedure using the Guelph System [9] in two ex-
by the moment of FTAI (P < 0.05), without interaction (P¼ NS). The periments, but different results were obtained among both exper-
results are showed in Table 1. In general, fertility was improved as iments that were attributed mainly to the operator skill. In the
semen deposition was deeper and delayed, with the greatest present study performed in multiparous Corriedale ewes, we found
pregnancy rate obtained with intrauterine FTAI by laparoscopy that pregnancy rate obtained with transcervical insemination is
performed at 54 h from device removal (56.9%, 66/116). Regardless intermediate (42.3%, P¼ NS) in comparison with cervical (36.0%)
the time of insemination, pregnancy rate was greater for laparo- and intrauterine insemination (50.2%). The transcervical method
scopic intrauterine than for cervical insemination, and the trans- used in this study is a bit different because it consist of the
cervical method was intermediate (Table 1). For transcervical manipulation of the cervix with the fingers directly by vaginal ac-
insemination, pregnancy rate was greater when semen was placed cess instead of transrectal manipulation previously reported [17].
deeper than 4 cm into the cervix (60.0%, Table 2), achieving in this Fertility obtained with transcervical method depends on the
case similar outcome than intrauterine insemination by laparos- penetration into the cervical canal. This technique was more
copy (P¼ NS). Deposition of semen deeper than 6 cm through the effective when semen deposition was deeper than 4 cm into the
cervix was possible in 9.6% (10/104) of ewes, and only in 8 of them cervix. In this case, pregnancy rate was similar than intrauterine
(7.7%) the insemination was completely placed into the uterus. laparoscopic insemination, reaching 60.0% of pregnant ewes.
Frequency for depth of semen deposition achieved with trans- However, the negative finding is that the effectiveness of trans-
cervical insemination is showed in Table 2. Regardless the insem- cervical route to reach the uterus in multiparous Corriedale ewes
ination technique, overall results showed greater pregnancy rate seems to be low. Only in <30% of ewes semen was placed deeper
with FTAI at 54 h (52.5%, 136/259) than 48 h (36.6%, 116/317) than 4 cm into the cervix, and only in very few females (<10%) the
(P < 0.05). insemination was performed completely into the uterus. Cervical

Table 2
Depth of semen deposition achieved with transcervical insemination and pregnancy rate obtained with fresh semen (100
106 sperm cells) after FTAI in multiparous
Corriedale ewes.

<2 cm into the cervix 2 to <4 cm into the cervix 4 to <6 cm into the cervix 6 cm or intrauterine

No. of ewes/total ewes 12.5% 58.7% 19.2% 9.6%

(13/104) (61/104) (20/104) (10/104)
71.2% (74/104) 28.8% (30/104)
Pregnant/FTAI ewes 38.5% 34.4% 65.0% 50.0%
(5/13) (21/61) (13/20) (5/10)
35.1% (26/74)a 60.0% (18/30)b

a vs. b, P < 0.05.

34 R. Casali et al. / Theriogenology 103 (2017) 30e35

canal length, tortuosity and penetrability is highly variable be- Author contribution
tween animals, and it is influenced by breed, age, previous lambing,
among other factors [4,16,24]. No information is available about the R. Casali performed the experiment, analyzed the data and
cervical penetration and the use of this technique for insemination wrote the manuscript. A. Pinczak, F. Cuadro and M. Guillen assisted
in Corriedale ewes, a breed worldwide used for meat and wool with the experiment. A. Mezzalira approved the final version of the
production. This study contributes with novel information in the manuscript. A. Menchaca designed the experiment, supervised the
use of this method, showing that the efficiency for cervical pene- work, analyzed the data and corrected the manuscript.
tration in this breed is relatively low.
From a practical point of view, for large-scale FTAI programs in Acknowledgements
which 300 to 500 ewes are inseminated every day from 46 to 56 h
from device removal, laparoscopy seems to achieve the greatest The authors thanks Carlos Sencion and Nicolas Secco of
pregnancy rate using half of sperm dose normally necessary for Fundacion IRAUy for their technical assistance during the experi-
cervical insemination, and much less time required than for ment. The financial support for this study was provided by
transcervical insemination. The time consuming by the technique is Fundacion IRAUy, Uruguay. R. Casali has a scholarship as PhD stu-
even more relevant for FTAI programs when the moment of dent of the UDESC, Brazil. A. Menchaca is fellow of Sistema Nacional
insemination is affecting pregnancy rate and the window for de Investigadores (SNI, ANII) and PEDECIBA, Uruguay.
insemination must be shortened. Regarding our findings in this
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