The use of additional oxytocin hormones in reproductive methods began in the
1970s in Europe. In recent studies, attempts have been made to achieve more
successful pregnancies by adding oxytocin to semen rather than administering
oxytocin exogenously to females. In light of this information, this study
aimed to understand the effects of adding low-dose oxytocin hormone into ram
semen on the pregnancy rate and reproductive efficiency of sheep. Semen was
collected from five Chios rams. The ejaculates were pooled and
Ovixcell® was used as the diluent. The estrus was synchronized
using an intravaginal sponge (Chrono-Gest®) from 122 Chios
ewes. After the sponge was left in the vagina for 12 d, a 500 IU PMSG
injection was made. In every sheep, intramuscular (IM) injections were inseminated by
the intracervical method using an insemination gun 50 h after injection.
Pregnancy results were obtained by ultrasound examination without practice.
Lambing performance was recorded at delivery; 53 out of 122 Chios ewes were
inseminated with oxytocin hormone content, and successful pregnancy was
achieved in 90.56 % of them. In the insemination of the remaining 69
sheep, oxytocin was not added, and the rate of successful pregnancy was
76.81 % (
Artificial insemination in sheep is an important reproductive technology. With the help of artificial insemination, male studs with superior genetics could help yield a much higher number of offspring (Robertson and Rendel, 1950). However, among the most important reasons why artificial insemination is not widely used in sheep today is the high cost of artificial insemination along with low pregnancy rates which could be considered unsuccessful (Olivera-Muzante et al., 2011; Lu et al., 2020). Achieving successful pregnancy rates would increase artificial insemination in sheep.
Many factors affect the impregnation of animals through artificial
insemination. Among these factors – both physical and environmental – are
species, breed, body condition score, number of lactations, milk yield,
environmental temperature, uterine tone, insemination time, insemination
operator, semen quality, reflux of semen, vaginal mucus and insemination
depth (Cameron et al., 1986; Anzar et al., 2003; Kershaw et al., 2005).
Optimizing the environmental effects could improve physical conditions. In
order to ensure sufficient sperm accumulation in the uterus, the cervix
needs to be passed at a certain level, which is one of the most important
activities in achieving a successful pregnancy (Olivera-Muzante et al.,
2020). Some factors may affect the progression of spermatozoa in the
reproductive tract; these factors could be classified as mechanical (folds,
crypts, cilia), cellular, physicochemical (vaginal secretion, cervical
mucus) and hormonal factors (Çoyan, 2005; Ömür, 2014).
Successful pregnancies cannot be measured solely by the success of forward
motility of spermatozoa; the effects originating from the female are equally
important. Several studies report the positive effect of insemination depth
on success (Eppleston et al., 1994; Falchi et al., 2021; Gutierrez et al.,
2022) – insomuch that being able to leave semen 1 cm forward
affects the rate of a successful pregnancy by 7 %–12 % (Eppleston et al.,
1994). The initial position of the semen released (pushing the semen forward
or backward) and the condition of the vaginal mucus during the initial
moment is also very important (Özmen and Cirit, 2020). The fluid in the
vagina during estrus is called cervical mucus. Particularly in sheep, the
amount, as well as the chemical and physical structure of vaginal mucus,
affects the semen passing the cervix (Linford, 1974; Özmen and Cirit,
2020). In addition, the contractility of the female reproductive tract
directly affects the result. The level of secreted prostaglandins and
oxytocin, the hormone of the posterior pituitary lobe, determines the
increase in the contractility of the reproductive canal. Increased uterine
contractility facilitates the progression of spermatozoa (Hawk, 1983; Kunz
et al., 1996; Gündoğan and Uçar, 2003; Suarez and Pacey, 2006;
Miki and Clapham, 2013; Ömür, 2014; Akthara et al., 2021).
Additionally, these contractions help remove the dead spermatozoa from the
uterus (Gündoğan and Uçar, 2003). Senger (1999) has reported
that prostaglandins (PGF2
Physiologically, the female sheep species' cervix structure is narrow, curved and sensitive (İleri et al., 2002). With folds much less than 1 cm, it is structured in a way to mislead the insemination gun (İleri et al., 2002; Lu et al., 2020). This structure makes it difficult to deliver semen inside (Deligiannis et. al, 2005; Kershaw et al., 2005). With the intracervical insemination method, it is observed that semen accumulates either in front of or slightly inside the cervix (Casali et al., 2017), which makes it difficult to meet with the oocyte. In order to address this problem, insemination could be done using laparoscopic methods. With the laparoscopic method, the aim is to enter surgically a place near the middle of the horn uterus, leaving the semen in the uterine cavity to ensure sufficient accumulation. This way, a direct transition is provided without ever touching the cervix (Kulaksız and Arı, 2016). Although laparoscopic insemination in sheep yields much more successful pregnancies compared to intracervical insemination, many factors affect both cost and success. It can be said that the laparoscopic insemination rate is lower due to the large number of equipment necessary, the high cost of the equipment, the need for surgical intervention and an experienced operator, and the stress on the animal (Kulaksız and Arı, 2016; Vallejo et al., 2019).
The intracervical method has been the most commonly used method for
artificial insemination in sheep until now (Fernandez-Abella et al., 2003;
Olivera-Muzante et al., 2011; Lopez-Perez et al., 2012; Galarza et al.,
2020; Abadjieva et al., 2020; Madrigalia et al., 2021). Although the rate of
successful pregnancies is relatively low in this method, it is preferred
mainly because, compared to the laparoscopic method, it costs less, requires
less experience and does not necessitate surgical intervention. Given the
fact that the speed of spermatozoa in intracervical insemination is 80–100
The use of additional oxytocin hormones in reproductive methods began in the 1970s in Europe (Duzinski et al., 2013). In recent studies, attempts have been made to achieve more successful pregnancies by adding oxytocin to semen rather than administering oxytocin exogenously to females (Pena et al., 1998; Langendijk et al., 2003; Duzinski et al., 2013; Okazaki et al., 2014; Kandemir et al., 2017; Manjarin, et al., 2019; Lu et al., 2020). While these are being done, the effects of the antioxidant property of oxytocin should be investigated. The antioxidant effect of oxytocin should be examined after semen is stored for long periods or after freezing and thawing. Examination of these effects in ram semen will guide future research. However, this research aimed to understand the effects of adding low-dose oxytocin hormone into ram semen on the pregnancy rate and reproductive efficiency of sheep.
This study was conducted in October, during the breeding season of sheep,
and lamb births took place in March. The research was achieved in the city
of Izmir, located between 39
Analysis results of feed given to ewes and rams. DM: dry matter. CP: crude protein.
Semen was collected from five Chios rams with the aid of an artificial
vagina (Minitube®, Germany). Semen characteristics include a volume
of 1.1–2.1 mL semen with semen density
To ensure oestrous synchronization, intravaginal sponges (Chrono-Gest®, Germany) containing hormones were inserted into all of the sheep used in the study. The sponges remained in situ for 12 d, and immediately after sponge removal, 500 IU PMSG (Chrono-Gest®, Germany) IM was administered.
Intracervical artificial insemination of sheep was done between 50–55 h
after sponge removal. Speculum and source of light were used during
insemination. The semen, containing
During the evaluation of the factors affecting the insemination results, the groups were not arranged randomly but formed according to the status encountered during insemination; i.e. if a small amount of fluid was encountered in the vagina during insemination, the group was classified as little; if there was a large amount of fluid, it was classified as large; and those that had a moderate amount of fluid were included in the moderate cervical mucus fluid group. Similarly, groups were classified according to the insemination depth results obtained. Inseminations were carried out in such a way that they could enter through the cervix with an insemination gun as much as possible; the classification of the groups was made as intracervix-2 for those with an entry of approximately 2 cm, intracervix-1 for those with an entry of approximately 1 cm, and as anterior cervix for those with no cervix entry. An insemination pipette with a different colour and with the dimensions on its tip was used when entering the cervix. This tip is 1 cm long and is in shades of blue. This straw allowed us to measure inflows in sheep with a lot of cervical mucus and a bit of manual pressure under the abdomen. Another feature investigated was the reflux of semen behaviour in the uterus. If absorption behaviour occurred after the semen was released immediately after entry with the insemination gun, the group was defined as non-rebounding; if no absorption behaviour was observed, it was defined as rebounding.
A pregnancy examination was performed 46 d after artificial
insemination. Pregnancy was diagnosed by ultrasonography. B-mod real-time
ultrasound with a 3.5 MHz probe (Mindray DP-50 Vet) was used. Those with at
least one fetus were defined as pregnant. The features examined in the
research, birth weight and gestational age were recorded after delivery.
pregnancy rate multiple birth rate lambing rate number of lambs at birth litter size female lamb rate male lamb rate
Data are presented as mean, and
The study investigated the effects of semen diluent (containing or not containing oxytocin) on reproductive performance. Pregnancy rate, multiple birth rate, lambing rate, litter size, female lambing rate, male lambing rate, and birth weights of lambs were measured in order to evaluate the reproductive performance. Table 2 illustrates the effects of semen fluids containing oxytocin and no oxytocin on reproductive performance.
Reproductive performance values of sperm fluids containing oxytocin and no oxytocin.
In the examination of the pregnancy rate, one of the indicators of
reproductive performance, the difference between the groups is statistically
significant (
The results of the semen rebounding behaviour depend on the amount of vaginal
mucus, insemination depth and uterine contractility, which is thought to
affect the insemination results, as given in Table 3. The amount of vaginal
mucus during insemination was found to be statistically significant in
achieving successful pregnancy (
Values of factors affecting insemination results.
In line with the aim of the study, it was determined that in Chios sheep whose heat was synchronized with exogenous reproductive hormones, the amount of oxytocin hormone added to the semen diluter had a high positive effect on success even with monochronic insemination. A total of 53 out of 122 Chios ewes were inseminated with oxytocin hormone content, and successful pregnancy was achieved in 90.56 % of them. Several studies report that the oxytocin hormone has been used to achieve successful pregnancy from the past to the present. In these studies, some researchers added the hormone into the semen, while others administered it to the animal intramuscularly (IM). Similarly to this study but in different animal species, added to semen, in their study on 1373 pigs, Hüln et al. (1977) reported that the results of insemination with the hormone oxytocin added to the semen were superior to those of the control group. Fuchs et al. (1989) reported that the use of oxytocin in cattle semen increased uterine contractions and the forward transport of spermatozoa. In their study conducted during the summer months, Pena et al. (1998) reported that a 77.02 % pregnancy rate obtained with oxytocin hormone added into pig semen, compared to the 54.39 % pregnancy rate obtained in the control group, was statistically significant. In their 2-year study on pigs, Duzinki et al. (2014) reported that based on 10 486 insemination results, semen with added oxytocin significantly increased pregnancy rates in all seasons. Okazaki et al. (2014) performed insemination with both fresh and frozen semen with the hormone oxytocin added to the porcine semen diluter. They reported results of 87.50 % in the oxytocin group, 70.50 % in the control group in the inseminations performed with fresh semen; in those performed with frozen and thawed semen, the results were 89.80 % in the oxytocin group, and 75.00 % in the control group. In their study of 800 inseminations in pigs 2 years in a row, Manjarin et al. (2019) reported positive results of oxytocin addition into the semen. This study and other research show that the oxytocin hormone added to the semen diluter helps transport sperm to the oviduct in the uterus. Similarly to this study, in the same animal species, Lu et al. (2020) used a diluter containing oxytocin in the insemination of 199 Kazakh sheep with oestrous synchronization. The pregnancy test results of the group receiving diluent with oxytocin were 85.50 %, whereas the result of 68.80 % was obtained in the control group. Researchers reported that the difference between these groups was statistically significant. These studies' results are congruent with those obtained in this study. The oxytocin hormone added to the semen may increase contractions as it progresses in the uterus, which would help the spermatozoa to move forward faster. As such, more spermatozoa will reach the female reproductive cell, possibly increasing the rate of successful pregnancies.
Many factors affect the achievement of successful pregnancy in artificial
insemination in sheep. Factors such as semen density, diluent, insemination
time and semen storage are among the most important. In studies conducted on
sheep at different times, it was reported that pregnancy rates between
11 % and 55 % were obtained in insemination with fresh semen. Olivera et al. (2005) reported that in inseminations performed with spermatozoon
density of
The study was conducted using the intra-cervical method with a speculum and light source. The vaginal mucus is seen before artificial insemination was classified based on the amount. In those sheep with a high amount of vaginal mucus, the pregnancy rate was determined as 20.00 %. However, in sheep with moderate and low levels of vaginal mucus, the success rate was determined as 64.29 % and 87.76 % respectively. Many studies have examined the ideal time for insemination in order to achieve successful pregnancies. The ideal time for insemination time is associated with the general condition of the vaginal mucus (Murtaza et al., 2020). Mahmoudzadeh et al. (2001) reported that the amount of vaginal mucus does not affect the success of insemination in cattle. However, Abril-Parreno et al. (2021) reported that the amount of cervical mucus in different sheep varies according to their breed and that the amount alone cannot be determinative. The present study finds that a high amount of vaginal mucus in Chios sheep negatively affects insemination success. The high amount is thought to increase the rate of ejection from the vulva due to the mass movement of the semen released into the vaginal mucus. Hence, it is thought that the rate of successful pregnancies is higher in sheep with a low or moderate amount of vaginal mucus.
As per the findings obtained in the research, oxytocin hormone added to ram semen diluter significantly increases the rate of successful pregnancy in sheep. Additionally, it was found that the amount of vaginal mucus during insemination adversely affects the success of insemination. In order to achieve a successful pregnancy, it will be beneficial to remove excess vaginal mucus with the help of an aspirator during insemination in sheep. In light of all these findings, it would be beneficial to conduct studies involving insemination in higher numbers in different breeds to better understand the effect of the oxytocin hormone on pregnancy.
The data used and analyzed during this study are available from the corresponding author upon reasonable request.
The author has declared that there are no competing interests.
Permission from the ethics committee regarding the animal experiments was received for this study (Republic of Türkiye Ministry of Agriculture and Forestry). The experiment was carried out according to all ethics and animal rights (DRC) considering all regulations in conformity with the European Union Directive for the protection of experimental animals (2010/63/EU).
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This paper was edited by Joachim Weitzel and reviewed by two anonymous referees.