Transcervical Insemination Research Articles

Reproductive Performance Following Transcervical Insemination with Frozen Thawed Semen in Ewes Submitted to Surgical Incision of Cervical Folds (SICF): Comparison with Laparoscopic Artificial Insemination.

Simple SummaryThe anatomical barriers of the female reproductive tract and the low quality of the frozen thawed semen of rams are limiting factors in spreading superior genotypes in the ovine species. Artificial insemination (AI) is therefore mostly performed by laparoscopy. The surgical incision of cervical folds (SICF) has been demonstrated to allow transcervical intrauterine semen deposition. Here we present data regarding pregnancy (PR) and lambing (LR) rates obtained by transcervical AI following SICF, compared to those obtained by laparoscopic AI using frozen thawed semen. The results showed that in 89.7% of ewes submitted to SICF intrauterine, the deposition of semen was allowed. Moreover, in both experimental groups, the PR and LR were similar. These observations suggest that SICF could represent a valid preliminary procedure to allow transcervical AI in animals of superior genotypes contributing to genetic improvement.Transcervical artificial insemination (AI) after the surgical incision of cervical folds (SICF) could represent a valid alternative to laparoscopic AI when frozen thawed semen is used. The aim of this experiment was to compare pregnancy (PR) and lambing rates (LR) of ewes submitted either to transcervical AI after SICF or to laparoscopic AI using frozen thawed semen. Pregnant at term ewes (n = 80) were allocated in two experimental groups. After lambing, one group (n = 39) was submitted to SICF. The remaining ewes that were regularly lambed were allocated to the group of laparoscopic AI (n = 40). Six months later, oestrous cycle of both experimental groups was synchronised and all ewes were artificially inseminated with frozen thawed semen. Ewes submitted to SICF underwent transcervical insemination and intrauterine deposition of semen was recorded. The remaining animals were submitted to laparoscopic AI. Pregnancy and LR were recorded. Intrauterine deposition of semen was possible in 89.7% pf ewes submitted to SICF. This group showed similar PR and LR compared to the laparoscopic group (respectively: PR, 71.8% vs. 70% and LR, 64.1% vs. 65%; p > 0.05). Transcervical AI after SICF may represent a valid alternative to laparoscopy in AI protocols requiring the use of frozen thawed semen.

PSX-12 Comparison of transcervical and intracervical artificial insemination techniques for fixed-time artificial insemination (FTAI) in the goat

Abstract The efficacy of transcervical (TC) and intracervical (IC) semen deposition was evaluated for pregnancy per artificial insemination (P/AI) in a fixed-time artificial insemination (FTAI) program in the goat. Three breeds (Alpine, n = 50, Boer, n = 17 and Spanish n = 20) were synchronized by a Co-synch program and then inseminated by TCAI (n = 52) or a commercial ICAI (RamGo, TecnoGen) technique (n = 35). On d0, the goats received a CIDR insert and a single injection of GnRH. On d12, CIDRs were removed and a single injection of PGF2α was administered, followed seventeen hours by an injection of GnRH. On d14, 49–50 hours post CIDR removal, the does were inseminated with fresh, cooled (4OC) semen. For TCAI, the external os cervix was visualized and the insemination gun inserted. Following manipulation through the cervical rings, semen was deposited in the uterine body. For ICAI, the straw was loaded into a RamGo intracervical catheter (ICC) and mixed with media to increase the volume. The doe was held at 60–70 degrees (head down). The external os cervix was visualized, the tip of the ICC placed in the os cervix and manipulated into the first cervical ring. Semen was deposited intracervically. Pregnancy status was determined on day 30–35 via transrectal ultrasound. No difference in P/AI was noted between the TC or IC technique (45.7 and 30.8 %, respectively). Additionally, no interactive effect on P/AI was found between insemination method and breed. The timing of FTAI did present a significant difference in the P/AI among the breeds. Spanish does demonstrated a lower P/AI than the Boer and Alpine does (10 vs 41.2 and 46 %, respectively). In summary, ICAI requires less technical proficiency and is a practical technique for FTAI in the goat. The P/AI results further demonstrate a need for continued evaluation of the optimal timing of insemination between breeds.

Welfare and pregnancy rate of ewes undergoing transcervical artificial insemination with ketamine subarachnoid anesthesia.

This study aimed to evaluate the impact of subarachnoid anesthesia with ketamine during transcervical artificial insemination (TCAI) on the welfare of ewes and on subsequent pregnancy rates. Ninety Suffolk adult ewes were randomized into three treatment groups: control group (CG), which underwent the TCAI procedure as established by cervical traction (CG; n = 30) and two groups that received subarachnoid anesthesia with ketamine at a dose of either 0.75mg/kg (KE0.75; n = 30) or 1.5mg/kg (KE1.5; n = 30) 5min before the cervical traction procedure. Intrauterine insemination was performed using frozen semen from three males previously analyzed and approved for fertility. The use of subarachnoid anesthesia decreased ewes' vocalizations (P = 0.0001) and abdominal contraction (P =0.0150) during cervical manipulations. The CG had more groans and vocalizations at the moment of cervix clamping and applicator passage through the cervix (P = 0.001). The cervix traction was facilitated by anesthesia. For the control group, most of the cervical traction was done just up to the middle of the vagina (P = 0.0021). Pregnancy rates increased significantly with anesthesia (P = 0.04) as shown by the rates of 40.0%, 56.7%, and 66.7% for CG, KE1.5, and KE0.75, respectively. The CG showed behaviors associated with absolute immobility, which is suggestive of distress. In brief, the use of ketamine in subarachnoid anesthesia for transcervical artificial insemination in ewes facilitated cervical traction, increased the pregnancy rate, and improved animal welfare.

Recent advances in cryopreservation of semen and artificial insemination in sheep: A review

Cryopreservation of semen facilitates widespread dissemination of superior germplasm and thus allowing the full exploitation of artificial insemination (AI) benefits. Recent focus is on preserving fertilizing capacity of frozen spermatozoa by improving the composition of extenders and changing cooling/freezing protocols, use of cryoprotectants, antioxidants, including enzymes, vitamins, amino acids and proteins, plant extracts and other compounds. In addition, research is also intensified on role of seminal plasma and its proteins as well as cholesterol-loaded cyclodextrin in semen freezing. Recently it has also been shown that helium-neon laser irradiation of cryopreserved ram sperm increased the post-thaw sperm motility, viability, cytochrome c oxidase activity and ATP contents with no significant changes in DNA and acrosome integrity. Post-thaw sperm motility of cryopreserved semen ranged from 33.3 to 72.2%. Fertility depends on the insemination technique and ranged from 45 to 80% for laparoscopic and 4 to 42% for transcervical insemination. Further, the complex anatomy of ewe's cervix is also a hurdle affecting intrauterine deposition of semen and consequently the fertility of frozen-thawed semen. Researchers are attempting cervical dilation with the use of hormones and chemicals to facilitate catheter penetration in the cervical canal. This review gives a detailed semen processing techniques and their effects on semen quality and AI techniques used in sheep and their success rates.

Surgery on cervical folds for transcervical intrauterine artificial insemination with frozen-thawed semen enhances pregnancy rates in the sheep

In sheep industry, genetic progress rate achieved by artificial insemination (AI) is limited by the convoluted anatomy of the cervix, which does not allow the passage of an insemination catheter for uterine semen deposition. The aim of this study was to test, in 98 pregnant at term Sarda ewes, the effects of: Experiment 1) total or partial ablation of cervical folds and Experiment 2) 4 or 2 incisions of cervical folds, on the passage of an insemination catheter, deposition of frozen-thawed semen and pregnancy rates. Surgical procedures were performed within 24 h from parturition providing deep sedation and epidural anaesthesia. Duration of surgeries and post-operatory recovery were carefully monitored. For both experiments, 5 months since surgery, independently of the stage of oestrus cycle, cervical patency was tested through the transcervical passage of a palpation probe. Six months since surgery, in Experiment 1, ewes were naturally mated with fertile rams. In Experiment 2, ewes submitted to incisions of the cervical folds and a control group underwent synchronisation of oestrus and transcervical AI with frozen-thawed semen. Thirty days later, for both experiments, pregnancy rates were assessed by ultrasonography and lambing rates were recorded. Five months after surgery, in Experiment 1, transcervical passage of a palpation probe to reach the uterine lumen was possible in all ewes submitted to total and partial ablation of folds. In Experiment 2, this was achievable in 90.5% ewes with 4 incisions of the folds and in 89.6% ewes with 2 incisions with no significant differences among groups (P = 0.44). In Experiment 1, pregnancy rates in ewes mated to rams after total or partial ablation of the cervical folds was 100%. In Experiment 2, following transcervical AI, pregnancy rates were higher in groups submitted to 4 (63.7%) or 2 (41.4%) incisions of the cervical folds compared to the control group (8%; P<0.05). These data were confirmed at lambing with rates of 56.8% and 41.4% in ewes submitted to 4 or 2 incisions respectively, significantly higher than the control group (4%; P<0.05). Surgical ablation or incision of the cervical folds in post-partum ewes represent valid procedures for transcervical intrauterine deposition of semen for AI, obtaining satisfactory pregnancy rates. These procedures might be useful in programs of genetic selection and MOET.

Morphometry and depth of inseminating catheter penetration in prolific and non- prolific ewes at different ages: A post mortem study

The ewe's cervix structure is highly variable among females, which may limit the application of transcervical artificial insemination (TCAI). This work aimed to study the variation in cervix anatomy in two different prolificacy breeds (D’man a prolific and Boujaâd a non-prolific sheep). Reproductive tracts were collected from D'man (n = 64) and Boujaâd (n = 187) females aged between 1 and 4 years. The weight of the uterus, length of the cervix and depth of penetration of the inseminating pipette were recorded. The number of cervical rings was counted and their arrangement graded. The cervix anatomy of D’man ewes differs from that of Boujaâd. The cervix morphology of Boujaâd ewes is more complex compared to D’man ewes; this complexity depends on the age of the ewe. No significant difference was found regarding the depth of penetration of the insemination catheter for the two breeds at 1, 2 and 4 years old (P > 0.05). However, at 3 years old, the highest depth of penetration (P < 0.05) was recorded in D’man ewes. In addition, for both breeds, grade 2 was the most frequent (P < 0.05), and the cervix became less complicated for TCAI with advanced age (4 years old). To conclude, the cervix of D’man ewes is less complex and more favorable to TCAI. In addition, to overcome the complexity of the cervix in Boujaâd sheep, a selection of 4-year-old ewes will make the penetration of the artificial insemination catheter possible.

Efeito da Aplicação de Flunixin Meglumine Sobre a Taxa de Gestação de Ovelhas Inseminadas via Transcervical

Background : : : : In livestock animals, which artificial insemination (AI) is widely spread, a healthy uterine environment is an important factor that should be taken into account for an adequate fertilization and establishment and development of pregnancy. The main limitation to spread the frozen semen insemination in sheep is the high costs of the laparoscopy AI and the difficulty of traversing the cervix. To overcome problems associated with transcervical artificial insemination, new techniques and instruments have been proposed. The manipulation of cervix during transcervical artificial insemination in sheep can lead to a local inflammatory reaction that releases inflammatory mediators, such as prostaglandins and cytokines, that either can negatively affect the uterine environment and consequently, the conception. In this regard, the injection of a non-steroidal anti-inflammatory (NSAI) at the moment of AI could reduce the secretion of inflammatory mediators, promoting a healthier uterine environment favorable to conception. The flunixin meglumine is a NSAI which reduces the synthesis of PGF 2 a by the inibition of the cyclooxigenase. The objective of this study was to evaluate the effect of flunixin meglumine (non-steroid antiinflammatory) injection on pregnancy rate of ewes inseminated by the transcervical technique. Materials, Methods & Results : In this experiment 69 pluriparous ewes were used, 29 in control group (CG) and 40 in flunixin meglumine group (FMG). The estrus detection was performed twice daily for 5 days. The ewes detected with paint in the low back were considered in estrus. The AI procedure was performed 8 hours after the estrus detection. At the AI time, the ewes were separated according to body condition score in two experimental groups. The ewes of the FM Group were injected with 1.1 mg/Kg of flunixin meglumine, im, 1 hour before the AI procedure. In order to locate the ostium, a vaginal speculum and a source of external light were used; whereas the fixation and traction of the ostium were performed with an Allis clamp, allowing the penetration of the semen applicator throw the vagina and cervix. The insemination procedure was performed 8 hours after estrous detection. There was no difference (P > 0.05) on the pregnancy rate between groups, whereas the Control group had 68.9% and the FM group had 60.0% of pregnancy. Discussion : The result demonstrates that the local inflammatory reaction caused by the traction of cervix was not detrimental to uterine environment and fertilization, since a high pregnancy rate was detected in both groups. Although the hypothesis of this study was not supported by the results, the high pregnancy rate detected in this experiment was higher than the results normally detected. Several studies have shown low pregnancy rates when transcervical artificial insemination with frozen semen is used associated to protocols of estrous synchronization. The flunixin meglumine did not improve the pregnancy rate, probably due to the insemination procedure adopted, since the traction of the cervix were performed gently, and consequently minimized the tissue damage. Thus, the inflammatory reaction may not have been sufficiently extensive as to lead to alteration of the uterine environment. These results suggested that flunixin meglumine injection did not influence the conception when was used prior to the transcervical insemination.

Influence of age on fertility rate and chicken hatchability at artificial insemination by semen diluted with VNIIGRZH extender

The ewe's cervix structure is highly variable among females, which may limit the application of transcervical artificial insemination (TCAI). This work aimed to study the variation in cervix anatomy in two different prolificacy breeds (D’man a prolific and Boujaâd a non-prolific sheep). Reproductive tracts were collected from D'man (n = 64) and Boujaâd (n = 187) females aged between 1 and 4 years. The weight of the uterus, length of the cervix and depth of penetration of the inseminating pipette were recorded. The number of cervical rings was counted and their arrangement graded. The cervix anatomy of D’man ewes differs from that of Boujaâd. The cervix morphology of Boujaâd ewes is more complex compared to D’man ewes; this complexity depends on the age of the ewe. No significant difference was found regarding the depth of penetration of the insemination catheter for the two breeds at 1, 2 and 4 years old (P > 0.05). However, at 3 years old, the highest depth of penetration (P < 0.05) was recorded in D’man ewes. In addition, for both breeds, grade 2 was the most frequent (P < 0.05), and the cervix became less complicated for TCAI with advanced age (4 years old). To conclude, the cervix of D’man ewes is less complex and more favorable to TCAI. In addition, to overcome the complexity of the cervix in Boujaâd sheep, a selection of 4-year-old ewes will make the penetration of the artificial insemination catheter possible.

Adding small ruminant reproduction to your practice

Equipment needs for transcervical insemination (TCI) of whitetail deer and goats are essentially the same. Trans-cervical insemination of sheep can use the same equipment, but pregnancy rates are low in this species with TCI, and the author does not recommend this technique for use in sheep, especially with frozen-thawed semen. Laparoscopic artificial insemination requires more equipment and some practice, but can be mastered with a little practice and patience. Collecting and freezing semen is not difficult, but highly technical and requires attention to detail. Most equipment is readily available at a reasonable cost. A list of the equipment needed and comments concerning the equipment are included in this presentation.

Semen deposition by cervical, transcervical and intrauterine route for fixed-time artificial insemination (FTAI) in the ewe

Semen deposition through the cervix into the uterus is a difficult technique in ewes and represents the main limiting factor for insemination in this species. The objective of this study was to evaluate the pregnancy rate achieved with a new transcervical insemination method in comparison with conventional cervical and laparoscopic intrauterine techniques. A total of 586 multiparous Corriedale ewes were synchronized for fixed time artificial insemination (FTAI) performed by cervical, transcervical, or intrauterine route at 46–50 h or 52–56 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), without interaction (P= NS). Overall, the fertility was improved as semen deposition was deeper and insemination was delayed. For transcervical insemination, pregnancy rate was intermediate (42.3%; P= NS) between cervical and intrauterine route (36.0% and 50.2%; P < 0.05), and was greater for those ewes inseminated beyond 4 cm into the cervix (60.0% versus 35.1% for insemination beyond or within 4 cm 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 inseminated 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.

A retrospective clinical study of endoscopic-assisted transcervical insemination in the bitch with frozen-thawed dog semen.

Since the conclusion of data collation from previously published work, a further 352 inseminations using frozen-thawed dog semen by endoscopic-assisted transcervical insemination (EIU) have been performed by the author. Insemination was performed on the second day in which crenulation of the anterior vagina was detected in conjunction with a progesterone concentration of >10ng/ml. All semen samples were analysed for total number of sperm, total motility and progressive motility using computer-assisted semen analysis (CASA). The insemination dose was based on the progressively motile normal spermatozoa (PMNS). Insemination was performed on all bitches as previously described using a ureterorenoscope. Additional extender was inseminated subsequent to the semen to expand and fill the uterus. The semen and additional extender were inseminated slowly over a period of 15-20min. Pregnancy was determined by B-mode ultrasound equipped with a 7.5-MHz probe whilst standing and/or via the whelping rate. The number of sperm inseminated ranged from 9×106 PMNS to 519×106 PMNS, with progressive motility values ranging between 20% and 80%. The overall pregnancy rate was 68% (238/352). When stratified by PMNS, pregnancy rates were as follows: >150×106 PMNS - 76% (110/145), 100-150×106 - 68% (87/128) and <100×106 PMNS - 52% (41/79). Pregnancy rate was significantly higher when >150×106 PMNS (p=.003) or 100-150×106 PMNS (p=.027) were inseminated compared to <100×106 PMNS. These data are concordant with previous reports recommending the insemination of >150×106 PMNS to maximize pregnancy rate. These results indicate that one optimally timed EIU insemination results in similar pregnancy rates to previous publications of one optimally timed, or two or more non-optimally timed inseminations using the Norwegian catheter.

Determination of the normal reference interval for anti-Müllerian hormone (AMH) in bitches and use of AMH as a potential predictor of litter size.

Anti-Müllerian hormone (AMH) is a reliable endocrine marker of ovarian reserve in many species with extensive literature in both humans and cattle. However, there are no known hormonal predictors of ovarian reserve and potential reproductive performance in the bitch. A prospective cohort study was performed involving 155 intact bitches of various ages (range 1.2-7.6years) and breeds that were presented for routine breeding management over a one-year period. All bitches were artificially inseminated with frozen or fresh semen using the transcervical insemination (TCI) technique. AMH concentrations were measured using a commercially available canine AMH ELISA (Ansh Labs® , Texas, USA), which we validated prior to performing the study. The reference interval (RI) for AMH for all bitches in the study, regardless of body weight, was 2.9-21.1ng/ml. There was a significant effect of bitch size and age on AMH concentrations. The RI for giant breeds was significantly (p<.01) lower (1.75-15.6ng/ml) than small-sized (5.6-24.2ng/ml), medium-sized (4.3-23.7ng/ml) and large-sized (4.3-21.0ng/ml) bitches. The mean AMH concentration in bitches less than 4years of age was 12.4ng/ml, whereas the mean AMH concentration in bitches older than 4years of age was 10.5ng/ml (p<.05). For each additional year of age above 1year of age, AMH concentrations fell by 0.5ng/ml. There was no effect of AMH concentration on the whelping rate. Smaller breeds had smaller litters (and higher AMH concentrations), but within each breed size category, bitches with higher AMH concentrations had significantly larger litter sizes (p<.01). For each 1ng/ml increase in AMH, litter size increased by 0.3pups/litter. In conclusion, we determined a normal reference interval for AMH in bitches based on body weight using a canine-specific assay. In agreement with findings in humans and other species, we found that there is a decline in AMH concentrations with advancing age in bitches. Finally, the significant positive correlation between AMH concentrations and litter size indicates that AMH may be a useful management tool for the selection of bitches in breeding programmes.

Assessing the usefulness of prostaglandin E2 (Cervidil) for transcervical artificial insemination in ewes

The underlying theme of this study involved the evaluation of the dilatory effects of prostaglandin E2 on the ovine cervix and thus the assessment of its potential applicability to transcervical artificial insemination (TCAI) in ewes. A novel method of prostaglandin E2 administration (controlled slow-release vaginal inserts) was examined, and the practical implications of this approach including cervical penetrability and posttreatment pregnancy rates were evaluated. The Guelph method of TCAI was performed during the seasonal anestrus (n = 40) and the breeding season (n = 40) on multiparous Rideau Arcott × Polled Dorset ewes, with or without the pretreatment with Cervidil (for a duration of 12 hours or 24 hours before TCAI). Cervical penetration rates averaged 82.5% (66 of 80), and they varied neither (P > 0.05) between the two seasons nor between Cervidil-treated ewes and their respective controls. Cervidil priming significantly reduced the total time required for TCAI during the breeding season in comparison with controls (54 vs. 98 seconds), especially after the 24-hour exposure (38 vs. 108 seconds). The time taken to traverse the uterine cervix was negatively correlated (P < 0.05) with the breed (percentage of Rideau Arcott genotype) and lifetime lamb production in seasonally anestrous ewes. Four out of 36 (11%) successfully penetrated ewes in the breeding season (three ewes allocated to the 12-hour control group and one ewe that had received Cervidil for 12 hours) became pregnant and carried the lambs to term. Vaginal mucus impedance at TCAI was significantly and positively correlated with the total time required to complete the procedure in cyclic ewes, and the negative correlation between vaginal mucus impedance and total time values at the time of controlled intravaginal drug release device removal approached to significance in anestrous ewes. The present results indicate a moderate benefit of using Cervidil for inducing cervical dilation before TCAI in ewes, mainly in the breeding season. The specific reason(s) for impaired fertility after the TCAI using frozen–thawed ram semen remains to be elucidated.

RESULT OF ADVANCED TECHNIQUES FOR ARTIFICIAL INSEMINATION IN EWES WITH FROZEN-THAWED SEMEN

In all domestic species, acceptable pregnancy rate with frozen-thawed semen is 20-30% through simply cervical and vaginal insemination. Researchers have proved that achievable high results can be expected only from intrauterine insemination in ewes. Unlike the cow, and goat, the cervix of ewes is a formidable barrier to penetrate for transcervical intrauterine insemination. It is not only due to the physical size of the canal and a narrow luminal diameter (even at estrus), but also due to its caudally facing eccentric series of four to eight funnel-like rings. The major aim of this study was to evaluate a comparison of transcervical and laparoscopic intrauterine artificial insemination techniques with frozen –thawed semen on the reproductive performance of ewes.58ewes of 3-4 years of age, so called Altanbulag subspecies of breed were selected and equally divided into 2 groups for the AI methods from October 15-20, 2013 in the breeding station of Research Institute of Animal Husbandry in Hongor sum, Darhan uul province. The estrus was synchronized with EAZI-BREED TMCIDR in which the progesterone content was 0.3 g. After 12 days, the CIDR were removed, PMSG of 400 and 200 IU were administrated intramuscularly per ewe. The total success of synchronization rate was 93.3%. The results has shown the possibilities of laparoscopic and transcervical AI techniques in 55.17%, 41.37% (p&gt;0.05) of pregnancy rates respectively, with frozen-thawed semen in ewes by ultrasonography diagnosis.Mongolian Journal of Agricultural Sciences Vol.13(2) 2014: 43-47

First deliveries after estrus induction using deslorelin and endoscopic transcervical insemination in the queen

The present study consists of two distinct parts, experiment 1 and experiment 2. In experiment 1, 13 anestrous queens were treated with a 4.7-mg deslorelin subcutaneous implant to assess its effectiveness in inducing estrus in the domestic cat. Deslorelin is currently used for the reversible suppression of ovarian and testicular activity in dogs and cats and for estrus induction in the bitch. Estrus induction is also reported in the queen but never reported with a targeted study. All the queens showed a positive response to the induction protocol, and estrus was detected within an average of 5.0 ± 2.2 days after the implant placement in 13 out of 13 subjects (100%). Seven of 13 queens exhibited behavioral manifestations of estrus, and the mean number of follicles detected at ultrasound examination was 4.8 ± 1.6 per subject. In experiment 2, three of the queens previously treated with deslorelin for estrus induction were submitted to artificial insemination through endoscopic transcervical catheterization, a new nonsurgical technique for intrauterine sperm deposition. All of them (100%) were pregnant after insemination and they gave birth to healthy litters. The study, as a whole, proves the effectiveness of the 4.7-mg deslorelin subcutaneous implants in inducing estrus in the domestic cat and is, to our knowledge, the first study assessing fertility of the induced estruses. Moreover, it shows the effectiveness of endoscopic transcervical catheterization for artificial insemination in the queen.

Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination.

The present study was conducted to evaluate whether supplementation of semen extender with glutathione (GSH) can maintain the quality of frozen-thawed canine spermatozoa. Eighteen ejaculates were obtained from 5 dogs and placed in extender (20% egg yolk, Tris, citric acid, lactose, raffinose, antibiotics and 6.5% glycerol) containing 0 (control), 2.5, 5, 7.5 or 10 mM GSH. The samples were cooled to 4 C and then frozen in liquid nitrogen vapor. Motility parameters of the sperm were evaluated at 0, 1, 2, 3, 4, 12 and 24 h after thawing. Sperm motility was higher in the 5 mM GSH group than in the control or 2.5 and 10 mM GSH groups; this effect was observed at 1 to 24 h after thawing (P < 0.05). The 5 mM GSH group had a higher sperm viability index at 12 and 24 h after thawing compared with the other groups (P < 0.05). Acrosome integrity, evaluated at 4 h after thawing, was greater in two of the GSH-treated groups (5 and 10 mM) compared with the control. Lipid peroxidation (LP) levels immediately after thawing were lower in the 5 and 10 mM GSH groups compared with the control, while those at 12 h after thawing did not differ significantly. Frozen-thawed semen in the 5 mM GSH group was used for transcervical insemination of 4 bitches, resulting in delivery of 5 puppies from 2 bitches. These results indicate that supplementation of semen extender with 5 mM GSH was effective in improving motility, longevity and acrosomal integrity and inhibiting LP levels in post-thaw canine spermatozoa, without any adverse impacts on full-term development after transcervical insemination.

Transcervical artificial insemination in dogs and cats: review of the technique and practical aspects.

The technique of transcervical catheterization for artificial insemination has gained practical importance over the last 3 and 1 decades in bitches and queens, respectively. The vagina of both species has a thickening of the dorsal aspect called dorsal medial fold, which restricts the lumen of the paracervix, making catheterization of the cervix difficult both with manual and endoscopic techniques. Manual catheters have been used initially in the bitch and are now gradually being replaced by rigid endoscopy through the adaptation of human cystoscopes and ureteroscopes. Cystoscopes provide excellent imaging of the vagina, but cervical catheterization is more difficult due to the oblique 30° viewing angle of its telescope and may not be long enough to catheterize large size bitches. Ureteroscopes allow an acceptable view of the vaginal mucosa and offer advantages such as the possibility to catheterize the cervix of bitches of all body sizes, manipulate the cervical tubercle when the external cervical os is not visible, offer a better visualization of the cervix using the shunt (a large Foley catheter which allows insufflation and distention of the vagina). Feline cervical catheterization has been achieved with three different types of catheters; the last one, developed following careful anatomical studies of how the feline vaginal lumen changes during estrus, allows cervical catheterization thanks to digital manipulation of the cervix through the rectum.

Comparison of endoscopic-assisted transcervical and laparotomy insemination with frozen-thawed dog semen: A retrospective clinical study

The objective of this retrospective clinical study was to compare pregnancy rates obtained after the use of endoscopic-assisted transcervical catheterization (EIU) or laparotomy (SIU) for insemination of frozen-thawed dog semen. Healthy bitches from various breeds were inseminated with semen from multiple donors processed by different freezing centers. Data from 118 inseminations (78 EIU and 40 SIU) performed between 2009 and 2011 were analyzed. Insemination timing was based on vaginal cytology, serum progesterone concentrations, and vaginoscopy. A ureterorenoscope and a CH-5 Transcervical insemination catheter were used for EIU; 28 of the bitches in this group were inseminated twice with the second insemination less than 12 hours after the first. The numbers of live morphologically normal sperm (LMNS) were determined to characterize insemination doses. Overall, pregnancy rate was greater (P < 0.05) in the EIU group (65%) than in the SIU group (45%). Pregnancy rates were greater (P ≤ 0.06) when more than 100 × 106 LMNS were inseminated regardless of insemination method; the greatest pregnancy rate was observed in the EIU group when this insemination dose was used (38/49; 78%). There was no significant difference in pregnancy rate whether one (69%) or two inseminations (64%) were performed in the EIU group. Complications in the SIU group included anesthetic-induced bradycardia during surgery, significant postsurgery pain, seroma formation over the abdominal incision, and delayed wound healing. No complications were noted during or after insemination in the EIU group. In conclusion, these results support the use of EIU as a noninvasive alternative to laparotomy for insemination of frozen-thawed dog semen. In addition, use of more than 100 × 106 LMNS is also recommended for insemination.

62 EFFECTS OF VARYING GLUTATHIONE CONCENTRATIONS IN SEMEN EXTENDER ON THE QUALITY OF FROZEN–THAWED CANINE SPERM

Trans-cervical insemination (TCI) with cryopreserved semen offers a potentially effective approach for breeding canids with specific genetic traits, such as guide dogs for the blind. However, there are technical difficulties in canine sperm cryopreservation, such as the composition of semen extender. The aim of this study was to evaluate the effects of glutathione (GSH) as an antioxidant in the semen extender to improve the quality of frozen-thawed dog sperm. A Tris-egg yolk-citrate extender containing 15.7 mg mL–1 of TRIS, 8.8 mg mL–1 of citric acid, 14.1 mg mL–1 of lactose, 25.4 mg mL–1 of raffinose, 1% (vol/vol) antibiotics, and 20% (vol/vol) egg yolk in ultra-pure water was used as the base medium. Twelve ejaculates were collected from 7 dogs. Each ejaculate was divided into 2 to 5 aliquots and extended with base extender supplemented with 0, 2.5, 5, 7.5, and 10 mM GSH as first dilution. The extended semen was equilibrated for 3 h at 4°C. An equal volume of second extender was added to obtain a final concentration of 6.5% glycerol and sperm per milliliter. The sperm samples were loaded in straws and frozen at 6 cm above the surface of LN2 for 15 min in a styrene foam box and plunged into the LN2. The frozen semen was thawed for evaluation. The motility of sperm was estimated with a phase-contrast microscope and the motile patterns were classified into the following grades: progressively motile at a high speed (+++), progressively motile at a moderate and low speed (++), motile without progression (+), and immotile (–). Then, the sperm motility index (SMI) was determined from the following formula as described previously (Iritani et al., 1975), with some modifications: the percentage of (+++) sperm + the percentage of (++) sperm × 0.75 + the percentage of (+) sperm × 0.5. Sperm motility and the SMI were determined at 0, 1, 2, 3, 4, 12, and 24 h after thawing. Acrosome status was evaluated at 4 h after thawing. Lipid peroxidation (LP) levels at 0 and 12 h after thawing were used to examine the antioxidant ability of GSH. Trans-cervical insemination was carried out on 5 bitches to evaluate the fertility of GSH-treated sperm. The TCI were performed nonsurgically with a laparoscope and deposited 2 mL of semen through a catheter. Each bitch was inseminated 1 to 2 times during oestrus. Data were analysed using ANOVA with the Tukey-Kramer method. We found that the rate of (+++) sperm in the 5 mM GSH group was higher than that in the 0 mM group from 1 to 24 h after thawing (P &lt; 0.05). The SMI was higher in the 5 and 7.5 mM GSH groups than in the 0 mM group (P &lt; 0.05). There were no significant differences in the control and 2.5 and 10 mM GSH groups. Long-term survival was increased in the 5 mM GSH group. Acrosome integrity was higher in the GSH-treated group. The level of LP was lower in the GSH-treated groups at 0 h after thawing (P &lt; 0.05). Trans-cervical insemination with the 5 mM GSH-treated semen resulted in the delivery of 5 pups from 2 bitches. These results indicate that the cryopreservation with 5 mM GSH can improve the motility, viability, and fertility of frozen-thawed canine sperm by its antioxidant effects on the sperm membrane.

Use of flunixin meglumine in Santa Inês ewes submitted to laparoscopic and transcervical insemination

The objective of this work was to evaluate the use of flunixim meglumine in Santa Ines ewes submitted to artificial insemination (AI). Forty-four Santa Inês ewes were synchronized and inseminated at fixed times, by the transcervical or laparoscopic route, between 52 and 58 hours after sponge removal. The ewes were split into two treatment groups, to receive intramuscular injections of 2 mL of saline (control treatment) or 2.2 mg/kg flunixin meglumine (FM treatment), twice a day between days 9 and 19 after AI. The pregnancy diagnosis was performed by ultrasound 30 days after the AI. The chi-square test was used to compare the pregnancy rate between the control and flunixin meglumine treatment and between type of insemination, while the t-test, at 5% probability was used to compare the average weight and body condition, using the SAS statistical software. Flunixin meglumine was not effect in increasing the pregnancy rate in Santa Ines ewes undergoing laparoscopic and transcervical insemination.