Transcervical Insemination Research Articles

Evidence-based practice in canine artificial insemination.

A number of methods are currently used to predict the optimal date of insemination of the breeding bitch, particularly with the use of frozen-thawed canine semen which has a far shorter lifespan than fresh semen. Aside from confirming cytological oestrus, vaginal cytology is of no assistance in predicting the most fertile day(s) in a bitch; however, a neglected avenue of research suggests that vaginal cytology may be of great importance in confirming the days of optimal fertility retrospectively. Similarly, vaginoscopy provides clues as to the stage of a bitch's cycle but is inadequate as a sole determinant of her most fertile days. Nevertheless, vaginoscopy is useful to identify very late oestrus and the onset of dioestrus, as well as Stage I of labour (cervical dilatation). Due to variations in the rate at which circulating progesterone concentrations rise in individual bitches, the reliability of circulating progesterone concentrations for determining the optimal day(s) of insemination with frozen-thawed semen decreases as values rise. Moreover, progesterone assay results can vary widely due to extrinsic factors such as the time of blood sampling, sample storage conditions and the assay employed. Finally, this review investigates evidence surrounding various insemination routes and suggests that well-performed vaginal insemination, even with frozen-thawed semen, may be an acceptable approach for cases where transcervical insemination is impractical.

Transcervical artificial insemination in the brown brocket deer (Subulo gouazoubira): a promising method for assisted reproduction in deer

The present study aimed to test the efficiency of transcervical artificial insemination techniques with cervical immobilization (TCAI-CI) or cervical traction (TCAI-CT), associated or not with the use of oxytocin (OT) as a protocol for cervical dilation, in the brown brocket deer (Subulo gouazoubira). The study was carried out in a crossover design using four adult females in two replicates with an interval of 60 days. Estrus was synchronized with oral melengestrol acetate (MGA) associated with estradiol benzoate and sodium cloprostenol. TCAI techniques were performed from 18 to 24 h after estrus onset. All females received either an i.v. application of 50 IU of OT (G-OT, n = 4) or 1 mL of saline solution (G-Control, n = 4) 20 min before the TCAI procedure. The TCAIs were performed using frozen-thawed semen motility 40%, vigor 3, acrosome integrity 87%, membrane integrity of 95% and 13% of total post-thaw defects from the same batch. Behavioral estrus was observed in 100% of the females, in both replicates. It was achieved a 50% (4/8) success of cervical transposition with semen deposition in the uterine. Regarding inseminations, most of them (87.5%) were performed using the TCAI-CT technique, and the overall conception rate was 50%. Cervical transposition times (< 1 min) and TCAI procedures (~ 17 min) were considered satisfactory. Thus, the performance of the TCAI-CI and TCAI-CT techniques was successful, regardless of using OT as a cervical dilation protocol. This procedure is proposed as a method of choice for artificial insemination with greater applicability in different conservation centers, compared to more advanced reproductive biotechniques, and with a favorable impact on the conservation of deer species.

Factors Affecting Pregnancy Success in the Bitch Following Transcervical Insemination

Endoscopic-assisted transcervical inseminations (TCIs) have become increasingly popular. The aim of this retrospective clinical study was to evaluate data from the TCIs performed at our facility. We evaluated data from January 2018 through December 2021. This included 137 cases with fresh, 67 cases chilled, and 63 cases using frozen-thawed semen. All bitches underwent breeding management to determine the ideal breeding period. All semen samples were evaluated for total number of sperm, total motility, and progressive motility. Pregnancy was determined by B-mode ultrasonography about 4 weeks after the breeding. Litter size was determined by radiographs performed around the last week of gestation. The pregnancy rate was 83.21% for fresh, 67.16% for chilled, and 66.67% for frozen-thawed semen. There was a significant difference in litter size between fresh semen (6.82 puppies per litter) and both chilled (5.21 puppies per litter) and frozen-thawed (4.59 puppies per litter) semen (P < .05). There was no significant difference in litter size between chilled and frozen-thawed semen. There was no difference in pregnancy rates between clinicians performing the inseminations. Pregnancy rate was not different when sedation was used for the insemination (66.67%) compared to when sedation was not used (74.84%; P > .05). Performing 2 TCIs during the fertile period, regardless of the semen type, resulted in an increase of 6.6% in pregnancy rate (P > .05) and an increase of 0.7 puppies per litter, on average (P > .05). These results can be used to help guide recommendations for breeding clients on the best options to increase both pregnancy rate and litter size for their breeding.

Выбор оптимального времени и методы искусственного осеменения собак

We collected, analyzed, and summarized information on the optimal timing and methods of artificial insemination of dogs at present. Scientific articles published in the bibliographic databases such as e-library, Scopus, and ResearchGate were used for this review. It has been established that artificial insemination has significant advantages over natural insemination, including reliable long-term storage of sperm in cryobanks, the possibility of exporting and importing genetic material without transporting animals, and obtaining offspring from elite male producers from anywhere in the world, even after their death.&#x0D; The time of artificial insemination and natural mating is determined by the date of ovulation using direct (ultrasound visualization of the ovaries) or indirect methods (picture of vaginal smears, multiple measurements of blood progesterone concentration, etc.). In dogs, the egg cell ovulates immature at the stage of the oocyte of the first order. For this reason, it is recommended to inseminate them on the 2nd… 5th days after ovulation, when egg maturation ends in the fallopian tube - the formation of a second-order oocyte.&#x0D; For dog insemination, freshly obtained, diluted chilled, and cryopreserved sperm are used. When using freshly obtained and diluted cooled sperm, the method of choice is an intra-vaginal method of sperm administration, and frozen-thawed sperm requires transcervical intrauterine insemination under video endoscopic control.

Canine transcervical insemination: history and technique

Artificial insemination in the dog was first described nearly 250 years ago. Until the 1970s, the process remained predominantly in depositing semen in the cranial vagina. Initially, surgery was required to successfully deposit frozen–thawed semen in uterus. However, societal demands to eliminate the need for general anesthesia and surgery for breeding have led to development of reliable and successful transcervical insemination procedures. Common barriers to perform successful transcervical inseminations include equipment expense and developing skills. In addition to practice, adopting suggestions provided by the author will improve practitioner’s success.

Success of artificial insemination in Goats: An overview

Globally there is growing demand for small ruminant production especially, goats for meat and milk purposes to feed the ever-increasing human population. Earlier goat breeding was generally practised by natural mating; but now artificial inseminations (AI) with elite buck semen are well in demand for improving herd production. Success rates of AI in goats varied widely with type of semen used, oestrus synchronization, insemination method, region, breed etc. In goats, pregnancy rate is comparatively higher by AI with chilled liquid semen (71 to 78%) as compared to frozen-thawed semen (21 to 57%). Intracervical insemination is widely used in goats and easy to perform within 2 to 3 min with a success rate of 59.8% using chilled semen and 43.9% using frozen-thawed semen. The trans-cervical insemination is difficult to perform due to highly complex cervical anatomy of goats, hardly 78.57% using chilled semen and 53.12% success rates are achieved using frozen-thawed semen. The success rate through laparoscopy aided trans-uterine deposition of semen yields higher pregnancy rates (60 to 80%) as compared to vaginal and trans-cervical AI. But laparoscopic insemination involves elaborate equipment and special skills. Under Indian conditions, frozenthawed semen and intracervical insemination is an easier way to adopt in field conditions for AI in goats. Further, there is urgent need to standardize the semen freezing protocols and novel semen extenders which may be breed-specific to achieve higher conception rate in goats.

Effect of post-thaw supplementation of seminal plasma to the cryopreserved spermatozoa of Malabari bucks on in vivo fertility

The study was conducted to assess the effect of post-thaw supplementation of seminal plasma to the cryopreserved spermatozoa of Malabari bucks on in vivo fertility. The ejaculates collected from Malabari bucks of good (n=3) and poor (n=3) semen freezability were pooled separately and cryopreserved. After thawing, seminal plasma from good semen freezability bucks was supplemented in such a way that group I (GFSP) included thawed semen of good freezability with supplementation, group II (GF) included thawed semen of good freezability without supplementation, group III (PFSP) included thawed semen of poor freezability with supplementation and group IV (PF) included thawed semen of poor freezability without supplementation. After a period of incubation for 10 min. at 37°C, the semen from each group was used for transcervical insemination of Malabari does of proven fertility. The results showed that there was a significant improvement in the conception rate with post-thaw supplementation. Percent of animals kidded also differed significantly in the four groups. But, prolificacy did not differ in GF and GFSP.

Cervical duplication in dogs

Two maiden bulldogs with cervical duplication were presented for breeding management. Dogs were successfully impregnated via endoscope-assisted transcervical insemination (TCI) and had their litters via cesarean surgery. A common uterine body between 2 cervical openings and 2 uterine horns was noticed (with no other reproductive abnormalities) at surgery. Duplication of the cervix has apparently not been previously described in dogs. With TCI becoming a more frequently used method of breeding, it is probable that defects involving failed or incomplete fusion of the paramesonephric duct during embryological development will be more frequently observed by clinicians.

Repeated pregnancy in a bitch, with a diagnosis of extensive cystic endometrial hyperplasia, after the exact determination of the optimal time for breeding

A 6-year-old bitch was presented to the clinical examination with the aim of determining the optimal time for intrauterine insemination by frozen-thawed semen. The bitch had already had two litters in the past. Extensive cystic endometrial hyperplasia was diagnosed by an ultrasound examination in the bitch. The timing of the endoscopic transcervical insemination by a combination of ultrasonographic examination of the ovaries and determining the progesterone concentrations contributed to the successful conception in the bitch. The ultrasonographic examination of the ovaries represents a non-invasive method which allowed us to view the changes on the ovaries in real time and, thus, is a very suitable method for the specification of the ovulation time in bitches. Cystic endometrial hyperplasia is considered to be a possible cause of infertility. Nevertheless, the bitch repeatedly conceived and had larger litter sizes. Moreover, a considerable reduction in the number of endometrial cysts was observed after the third pregnancy of the bitch. In the described clinical case, the influence of the cystic endometrial hyperplasia on the conception was not demonstrated.

Artificial Insemination in Queens in the Clinical Practice Setting: Protocols and challenges.

Despite substantial advances in assisted reproductive techniques having been recently reported in cats, the use of these is limited and routine application is still far from being a reality in veterinary clinics. Nevertheless, there is an increasing demand from domestic cat breeders for artificial insemination (AI) techniques that are already commonly used in dogs. Where natural breeding is not possible in tom cats and queens of high breeding value, AI could offer a solution. Clinical challenges: AI in cats is more difficult than in other species - both in terms of semen collection/handling and oestrous cycle management given that ovulation must be induced. For practitioners wishing to perform AI in queens, there are challenges to overcome, and a good understanding of the techniques and procedures involved is pivotal. This review aims to contribute to improved knowledge by providing an overview of AI protocols, encompassing choice of breeding animals, procedures for semen collection, oestrus and ovulation induction, AI techniques and equipment. Depending on the animals involved and the specific AI technique chosen, essential equipment may include an artificial vagina, electroejaculator, endoscope (sialendoscope, which can be fairly expensive) and special catheters for transcervical insemination. Other instrumentation and materials needed are typically readily available in a veterinary clinic. In general, no particular skills are needed to perform the procedures described in this review, with the exception of endoscopic transcervical catheterisation, where the ability to use an endoscope is required. The information and advice/recommendations provided are based on specific feline research and reviews published in scientific peer-reviewed journals, animal reproduction textbooks, and presentations at national and international congresses. The authors also drew on their own clinical experience with regard to the choice of protocols and procedures presented in this review.

Effect of L-arginine and Eugenol on Ram Semen Kinematic Parameters and Post Thawed Fertility Rate after Trans-cervical Artificial Insemination

Backgroung: Long term storage of spermatozoa is one of the most desirable tools of assisted reproductive technologies. The purpose of this research was to evaluate the effect of two antioxidants on sperm kinematic parameters after the freezing-thawing process and the post thawed fertility rate after the trans cervical artificial insemination. Methods: Ejaculates from Lacune rams were collected using artificial vagina, were pooled and diluted using based extender (Biladyl, Minitube) and based extender with antioxidants (Eugenol, Arginine). Trans-cervical artificial insemination was performed with frozen seminal material in straws. Result: No significant differences were observed in samples cryopreserved in a base extender and in the base extender supplemented with Arginine. In Eugenol supplemented extender the kinematic parameters were significantly higher for p=0.5 (30.15% progressive motility after thawing) than in the others groups (23.57%, 24.38%). The fertility rate was higher for the straws conserved in extender supplemented with Eugenol (80%) compared with the base extender (55.55%) and the base extender supplemented with Arginine (60%).

Heterospermično osjemenjivanje u pasa

The idea of dual sire insemination in dog breeding is to give equal chances to both males to fertilize the eggs due to the specific oestrus cycle of the bitch. Two sexually mature, privately owned females Petit Basset Griffon Vendeen (bitch A and B) aged 3 to 5 years were subjected to dual-sire inseminations. Prior to insemination, the semen of stud dogs (n=5) was collected and evaluated for volume, concentration, total sperm count, motility, membrane integrity (HOS test), percentage of live spermatozoa and sperm morphology (eosin nigrosin staining). The time of insemination was based on serum progesterone (P4) concentrations, where P4 of &amp;gt;5–10 ng/mL was considered ovulation. Bitch A was inseminated in two oestrus cycles with fresh mixed semen from two males using the endoscopic transcervical insemination technique (TCI). Bitch B was inseminated in one oestrus cycle by laparoscopic intrauterine deposition of frozen thawed semen. Semen evaluation showed minimum deviations in all tested parameters between the chosen males on the days of insemination. Depending on sperm concentration and quality, the volume of ejaculates was adjusted, resulting in an equal number of motile spermatozoa from two males in each insemination, providing them an equal chance for fertilisation. Confirmation of pregnancy was carried out 24 days after insemination by ultrasonography. The whelping outcome was obtained directly from the owner of the bitches. Puppy blood was taken immediately after whelping from the umbilical vein into EDTA tubes. Blood samples were also obtained from the dam and both sires for DNA profiling. Parentage was determined for each dual-sired pup by using Thermo Scientific Canine Genotypes Panel 1.1. All inseminations resulted in pregnancy and whelping. A total of 14 puppies were born in three litters. Mixed parentage was determined in 1 of the 3 resultant litters (bitch A). In conclusion, dual sire insemination is a useful breeding tool providing the opportunity to obtain puppies from multiple genetic backgrounds in a single litter. However, as this method usually produces offspring from single father, optimal insemination protocol should be established for producing a litter of mixed paternity.

The use of oxytocin to cause cervical dilation for transcervical insemination in nulliparous goats: Improving pregnancy and kidding rates

To evaluate the effect of oxytocin as a cervical dilator, a study was carried out on nulliparous goats inseminated transcervically at the beginning of the breeding season. One hundred sixteen nulliparous goats with a mean live weight of 33.4±0.68kg and an age of 13.7±0.37months were used. The goats were exposed to active bucks of proven fertility for a period of 14 d in order to induce oestrus. One week later, the Ovsynch protocol was applied, which consisted of the application of 20mg of gonadorelin (Day Zero), 0.075mg of cloprostenol (Day 7) and of a second dose of 20mg of gonadorelin applied on Day 9. Artificial insemination (AI) was performed 16hr later. Three treatments were evaluated: T1=50IU saline, T2=25IU oxytocin; T3=50IU of oxytocin, intravenously applied 10-15min before AI. The time required to inseminate each treated goat from groups T2 and T3 was 49.56 and 56.25s, respectively, versus 85.78s needed for the goats from group T1 (p<.0001). In the T1 group of goats, the insemination catheter was inserted 2.1cm into the cervical canal and in goats from groups T2 and T3 it reached 3.41 and 3.77cm into the cervical canal, respectively (p=.02). Pregnancy rates and prolificacy (kids/doe) were higher (p=.02) for groups T2 (82.93%; 1.16) and T3 (76.92%; 1.21) respectively than for control goats (61.11%; 0.69). In conclusion, the intravenous administration of oxytocin led to greater dilation and depth of cervical penetration, obtaining higher pregnancy rates and prolificacy.

Factors Affecting Pregnancy Success in the Bitch Following Transcervical Insemination

Factors Affecting Pregnancy Success in the Bitch Following Transcervical Insemination

The Design of a New Catheter for Transcervical Artificial Insemination in Ewes.

Simple SummaryIn ovine species, transcervical artificial insemination is not easy to apply, due to the tortuous lumen of the cervix that does not allow the passage of routinely used catheters. Moreover, the quality of frozen–thawed semen in small ruminants is poor and these factors negatively affect the wide spreading of superior genotypes. The aim of this study was therefore to preliminarily test three newly designed insemination catheters, with bent tips of different lengths, in terms of reproductive performances in pluriparous ewes inseminated with frozen–thawed semen. Afterwards, the outcomes of insemination with the best performing catheter were compared to those obtained in ewes previously submitted to surgical incision of cervical folds, a technique that allows transcervical intrauterine deposition of semen. The results obtained indicated that a catheter with a bent tip of 5.0 mm allowed deep and fast intrauterine insemination, leading to pregnancy rates similar to those obtained following surgical incision of the folds. Further tests on the efficiency of the catheter are needed in field conditions and on a larger number of animals to assess the feasibility of the method in wide commercial insemination campaigns.In ovine species, transcervical artificial insemination (TCAI) is limited by the poor quality of frozen–thawed semen and by the convoluted cervical lumen hampering the passage of inseminating devices. The aim of the study was to test the efficiency of three newly designed catheters with bent tips of 3.5 mm, 5.0 mm or 8.0 mm in terms of reproductive performances (experiment 1) and to compare the results of TCAI with the best performing catheter of experiment 1 to those obtained in ewes submitted to surgical incision of cervical folds (SICF) prior to insemination (experiment 2). The following parameters were assessed: time to pass the cervix; depth of cervical penetration; site of deposition of semen; pregnancy (PR); and lambing rates (LR). The results of experiment 1 indicated that the 5.0 mm tip catheter resulted in deeper and faster TCAI and higher PR and LR compared to 3.5 mm and 8.0 mm tip catheters (p < 0.05). In experiment 2, TCAI with the 5.0 mm catheter did not differ from TCAI after SICF in terms of depth of semen deposition, time to pass the cervix, PR and LR (p < 0.05). In conclusion, the use of a catheter that allowed transcervical uterine deposition of semen without excessive manipulation led to satisfactory pregnancy rates.

Effectiveness of embryo transfer in cows - risk factors including in vivo derived and in vitro produced embryos

Abstract Multiple Ovulation Embryo Transfer is a biotech method with more than 50 years of history and an established position in cattle breeding. This procedure is beneficial in many ways, but it also carries a risk of failure. The study presents the overview of the most important risk factors that may affect conception rates in the course of embryo transfer, including the factors associated with the embryo sourcing (embryo production method, embryo quality, development stage and breed, embryo storage method), embryo transfer procedure (synchrony/asynchrony, embryo transfer difficulty, the time of the transcervical insemination gun passage, depth of embryo deposition, localization and structure of the corpus luteum relative to the follicle and both individual characteristics of donors and recipients (level of concentration of progesterone, the state of health of the udder, lactation level, body condition score and age) and some environmental factors.

Effect of Different Preservation Time of Chilled Semen on the Fertility of Field Indigenous Ewes

Background: Artificial insemination (AI), among all the fundamental systems of animal breeding, has proved to be the best and efficient method for the rapid improvement of livestock for maximum use of superior genetic merit of males on numerous dams. The frozen AI in sheep is, to date, not fulfilling the farmer’s need. Therefore, the present study aimed to determine the pregnancy rate in indigenous ewes with chilled semen preserved at different hours. Methods: Semen was collected from three indigenous rams using an artificial vagina and evaluated for its quality both in the fresh and chilled stage at 12 h, 24 h and 48 h before insemination. Trans-cervical insemination was performed in PGF2α synchronized ewes. Pregnancy was confirmed by ultrasonography at 50-60 days of gestation. Result: The motility, viability and normal sperm values of chilled semen decreased significantly (p less than 0.01) with increasing the duration of preservation. However, the quality of chilled semen was acceptable level for insemination. The pregnancy rate was significantly varied (p less than 0.01) and the higher pregnancy rate (64.28%) was achieved when AI was performed using semen stored at 4oC for 12 h than 24 h (58.33%) and 48 h (50%), respectively. Therefore, increased the duration of preservation time of different chilling process negatively affect the semen quality and the pregnancy rates.

Spermatozoa Obtained From Alpaca vas deferens. Effects of Seminal Plasma Added at Post-thawing.

The viscous seminal plasma (SP) is currently a major impediment to the handling of ejaculate and the development of some biotechnologies in South American camelids. The vas deferens-collected spermatozoa of alpacas is a useful technique to avoid this problem. On the other hand, SP contains a large protein component that has been implicated in the function of spermatozoa within the female reproductive tract. In this sense, the low fertility achieved using transcervical insemination with frozen-thawed spermatozoa in alpacas could be improved by adding SP. This study aimed to evaluate the effect of the whole SP on some in vitro parameters of alpaca spermatozoa after the freezing-thawing-process and the fertility after artificial insemination. It would contribute to a better understanding of the interaction between thawed sperm cells and SP. Spermatozoa were obtained by surgically diverted vas deferens. The samples were diluted with a Tris-based extender, packaged in straws, and frozen. At thawing, each straw was divided into two post-thawing conditions: with the addition of 10% of PBS (control) or with 10% SP (treatment). The sperm cells were evaluated using dynamic parameters, sperm cell morphology, and morphometry. Fertility was assessed by an artificial insemination trial. All in vitro parameters were analyzed by ANOVA. A heterogeneity test was scheduled for the fertility trial. After the freezing-thawing process, motility and plasma membrane functionality was improved when SP was added. No differences were found for post-thaw viability between the control and treatment samples. The percentage of normal cells was higher with SP at post-thawing, and a decrease of the presence of bent tailed spermatozoa with a droplet in the SP group was observed. The length of the head spermatozoa was 3.4% higher in the samples with PBS compared to those in which SP was added. Females pregnant at day 25 post-insemination were 0/12 (with SP inside the straw) and 1/10 (without SP inside the straw). In conclusion, the presence of 10% SP at post-thawing improves sperm cells' motility, functionality, and morphology, indicating that it would be beneficial to improve the frozen-thawed alpaca's physiology spermatozoa. More fertility trials must be developed to increase this knowledge.

Local α1-adrenergic blockers: An alternative for sheep cervix dilation?

To facilitate transcervical artificial insemination in sheep, the effects of local treatment with α1-adrenergic receptor antagonists on cervix dilation and hemodynamics were evaluated. Ewes (n = 7) were subjected to oestrous synchronisation every 40 days and assigned to treatments in a Latin square experimental design (seven animals × seven periods) with a factorial treatment arrangement (A × B), Factors A (prazosin or tamsulosin) and B (1, 2, or 4 mg/animal). Ewes of the six treatment groups (P1, P2, P4, T1, T2, and T4) were administered α1-adrenergic receptor antagonists while those of the control group (CG) were administered only α1-adrenergic antagonist carrier agent. Distance that the transcervical catheter penetrated without cervical resistance, mean arterial pressure, and uterine artery dopplerfluxometry were evaluated before and after 30 min, 1, 2, 4, 8, and 10 h of treatment. Catheter penetration distance was greater in ewes of the T4 and P4 groups (P < 0.01), with there being a positive correlation between dose and distance (r = 0.243). The penetration distance was similar (P = 0.84) for treated groups, with the greatest penetration occurring 2, 4, and 6 h after treatment (P < 0.01). The passage into the uterine lumen was greater (P = 0.013) in ewes of the P4 (17.9 %) and T4 (19.6 %) groups. There were no effects on blood pressure or uterine blood flow (P> 0.05). These preliminary results indicate there are benefits of treatment with 4 mg/animal of tamsulosin or prazosin in catheter passage through the sheep cervix 2–6 h after administration without hemodynamic effects.

Effects of cervical ripening treatments on pregnancy rates following transcervial artificial insemination in ewes

The study was aimed to identify the effective cervical ripening treatment to increase the pregnancy rates following transcervical artificial insemination (TC-AI) in indigenous ewes of Bangladesh. Three treatments schedules were compared, G1 (n = 6, control), G2 [n = 6, prostaglandin E1 (PGE1) as Misoprostol-Cytomis®] and G3 (n = 6, estrogen as Oestriol-Ovestin® + oxytocin as Linda-S® Vet). Ewes (n=18) were synchronized for estrus using two doses of 100 µg prostaglandin (PGF2α; Ovuprost®) 9 days apart. The cervical ripening treatments were administered just after the onset of estrus and left for 12 h before TC-AI. The cervical ripening was reflected with the depth of cervical penetration. The depth of cervical penetration in G1, G2 and G3 both before and after cervical ripening treatments were 0.33 ± 0.10 cm vs 0.43 ± 0.07 cm, 0.41 ± 0.09 cm vs 3.50 ± 0.26 cm and 0.43 ± 0.09 cm vs 1.54 ± 0.14 cm, respectively. The depth of cervical penetration increased significantly (p