Use of Hysteroscopy for Diagnosing Causes of Infertility in Camelids.

Hysteroscopy in alpacas and llamas allows for the identification of abnormalities on the surface or within the endometrium that cannot be identified with other methods. Hysteroscopy also allows for site-directed endometrial cytology, culture, and biopsy to achieve a definitive diagnosis. Even when no cause for infertility can be found, previously infertile females tend to become pregnant and maintain their pregnancies to term following the hysteroscopic procedure. This therapeutic effect may be a response to pre-hysteroscopy estrogen treatment, dilation of the uterine horns during hysteroscopy, and/or posttreatment uterine lavages. Complications following hysteroscopy have not been reported in camelids.

Lack of Efect of Short-term Lupin Grain Feeding on Ovulation Rate in Non-prolific Polish Mountain Ewes during the Breeding Season: Ultrasonographic and Endoscopic Assessment of Ovarian Activity.

The specific changes in antral follicle numbers and wave-like development have remained unrevealed in cyclic ewes fed high-protein, high-energy lupin grain for 6 days during the luteal phase of the estrous cycle (i.e., short-term nutritional flushing). This study was mainly conducted to determine ovarian effects of the 6-day lupin grain feeding in non-prolific Polish Mountain ewes, using transrectal ovarian ultrasonography and abdominal videoendoscopy. Estrus and ovulations were synchronized in 24 ewes with progestin-releasing intravaginal sponges for 12 days during the middle portion of the breeding season (September-October; 50.0458°N, 19.8406°E). Twenty-four ewes were assigned to three equal groups (n=8 each), including the Control group being fed the maintenance diet (i.e., hay-only), Treatment 1 receiving 500 g of lupin grain once a day, and Treatment 2 receiving 250 g of lupin grain twice a day, from days 9-14 of the synchronized estrous cycle (day 0=first ovulation of the interovulatory period studied). No differences were observed in the mean ovulation rate among the three groups of Polish Mountain ewes (P>0.05). Ovarian antral follicles emerging in the penultimate wave of the estrous cycle in Treatment 2 ewes had a longer growth phase (p <0.05) and attained a greater diameter (p <0.05) before ovulation, in comparison to those in the other two groups. A final wave of the interovulatory interval emerged ~1 day earlier in Treatment 2 than in Treatment 1 ewes (p <0.05). Nutritional supplementation with lupin grain increased the number of 3-mm follicles in Treatment 2 ewes (p <0.05). The results of this study indicated that short-term nutritional flushing with lupin grain from mid- to late luteal phase did not consistently enhance ovulatory responses in non-prolific genotypes of ewes. Although the administration of lupins altered the timing of wave emergence, ovulatory follicle diameter, or duration of different stages of the follicular lifespan, it failed to increase the number of ovulatory follicles emerging in the penultimate and final waves of the estrous cycle in non-prolific Polish Mountain sheep.

Identification of vessel degeneration and endometrosis in the equine endometrium, using narrow-band imaging hysteroscopy.

In this study, endometrosis and angiosclerosis in mares were studied. Endometrosis is a severe, progressive, and irreversible fibrotic condition that affects the endometrium, whereas angiosclerosis refers to thickening of vessel walls due to degenerative changes leading to reduced elasticity of the walls and lower perfusion. Histologic evaluations were performed on biopsies and compared with vascular features of the endometrial surface obtained via narrow-band imaging (NBI) hysteroscopy. First, it was determined if hysteroscopic evaluation of the endometrium using NBI resulted in a better visualization of the vascular pattern (i.e., vessel-versus-background contrast was increased) compared with using white light. This was found to be the case for examinations in vivo (n = 10), but not when using abattoir uteri (n = 3). In the second part of this study, it was determined if vascular densities and sizes as derived from NBI images could be used as indicators for the degree of degenerative changes of the equine endometrium and its vessels. Narrow-band imaging hysteroscopic evaluations were performed (n = 10), and endometrial biopsies (n = 32) were collected. Histologic specimens were evaluated for degree of endometrosis and angiosclerosis, and they were classified in Kenney categories. Narrow-band imaging images were analyzed for vascular pattern. Samples classified to Kenney category I, or without signs of vessel degeneration, had significantly higher vascular densities than samples from Kenney category IIa or with angiosclerosis. In conclusion, narrow-band imaging facilitates enhanced visualization of the vasculature of the equine endometrium during hysteroscopies, which has applications in detection of endometrosis and angiosclerosis.

Equine endometrial vascular pattern changes during the estrous cycle examined by Narrow Band Imaging hysteroscopy.

The aim of this study was to evaluate the uterine blood supply and endometrial vessel architecture, during the equine estrous cycle. Narrow Band Imaging (NBI) hysteroscopy was used for evaluating changes in the endometrial vasculature during the estrous cycle [six mares, d 0 (representing the day of ovulation), d 6 and 11 in four locations]. In addition, endometrial biopsy samples were used for immunodetection of markers for angiogenesis (Vascular Endothelial Growth Factor A, its receptor 2, as well as angiopoietin-2 and its receptor-tyrosine-kinase Tie2) during the estrous cycle (three mares, d 0, 5 and 10; one biopsy per mare). Detailed analysis of hysteroscopic images revealed an increase in the vascular density from estrus towards diestrus. In contrast, microscopic specimens prepared from biopsies revealed no evidence for changes in the endometrial vessel number during the estrous cycle. Studies on expression of angiogenesis markers indicated that cyclic changes in the endometrial vascular density observed by NBI-hysteroscopy were not due to formation of new vessels. It is concluded that vessels are involved in blood supply of a smaller area during diestrus, facilitating better distribution of nutrients during this phase.

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.

Endoscopic-assisted resection of a pedunculated uterine leiomyoma with maximal tissue preservation in a cow and a mare.

OBJECTIVE: To report successful minimally invasive treatment of a uterine leiomyoma in a cow and a mare. STUDY DESIGN: Clinical report. ANIMAL POPULATION: Limousine cow (n = 1), Thoroughbred mare (n = 1). METHODS: A 10-year-old cow and an 18-year-old mare were presented for difficulties in breeding and infertility, respectively. Examination of the reproductive tract revealed the presence of a large mass attached to the uterine wall via a wide and short peduncle in both cases. The mass expanded into the uterine lumen in the mare and into the abdomen in the cow. Both masses were removed using a minimally invasive endoscopic approach and a vessel-sealing and dividing device. RESULTS: Minimally invasive surgical resection of a subserosal and a submucosal leiomyoma with maximal sparing of uterine tissue resulted in a short convalescence period and apparent return to breeding function in a cow and a mare. Use of a vessel-sealing and dividing device provided excellent hemostasis and decreased tissue handling. CONCLUSION: Leiomyoma with short, wide, and thick peduncles were treated successfully in a cow and a mare with minimally invasive endoscopic approaches aiming at maximal uterine tissue preservation.

Successful use of endoscopy for transcervical cannulation procedures in the goat.

Two methods for transcervical cannulation of the goat were evaluated during a contraception study in 15 adult female Nigerian dwarf and African pygmy goats. Twenty-four transcervical cannulation procedures were conducted in which seven females underwent the procedure 2-3 times. Initially, a rigid 4-mm stainless steel cannula and external light source were used in 19 procedures to introduce the contraceptive compound into the uterus. Placement of the cannula was directed by feel or depth assessment. Of seven females that were euthanized following this procedure, four evidenced complications including penetration of the cervix with the cannula and cervical damage. A 2-mm custom-made endoscope with a specially designed cannula was then used for the remaining five procedures. No complications were found. A single animal, that underwent the endoscopic procedure twice, was euthanized for study purposes and no abnormal findings of the reproductive tract were reported. The use of an endoscope resulted in better outcomes because the uterus could be visualized after traversing the cervix.

Efficiency of hysteroscopic visualization of bubaline uterus.

Abattoir derived buffalo genitalia (n=98) were evaluated for uterine pathology employing palpable, ultrasonographic and hysteroscopic visualization. A high proportion (45.92%) of genitalia evidenced uterine pathology including endometritis (14.29%), pyometra (7.14%), hemorrhages in the endometrium (14.29%) and mucometra (6.12%). The sensitivity and specificity of hysteroscopy was 100% for endometritis and hemorrhages whereas for same conditions ultrasonography and palpation had low sensitivity and specificity. For pyometra and mucometra the sensitivity and specificity of ultrasonography and hysteroscopy was similar. It was concluded that for the diagnosis of minor pathological changes in the uterus such as endometritis and hemorrhages which can hamper fertility hysteroscopy is more efficient technique.

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 × 10(6) 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 × 10(6) LMNS is also recommended for insemination.

Hysteroscopy and episiotomy in a rescued, cold-stressed Florida manatee (Trichechus manatus latirostris) for diagnosis and treatment of a retained fetal skeleton.

A rescued female manatee was observed expelling a fetal bone from the vulva. The manatee was anesthetized and diagnosed with uterine retention of a fetal skeleton by ultrasound and hysteroscopy. Episiotomy was performed to gain manual access to the vagina and uterus for removal of the skeleton. Second intention healing of the episiotomy site produced excellent results. Rescued female manatees should receive a thorough reproductive tract evaluation since presence of retained fetal tissues might not be evident in blood or hormone analyses. Retention of a whole or partial dead fetus can be life-threatening to manatees, and retained tissues should be removed as early as possible.

Pregnancy rates in mares inseminated with 0.5 or 1 million sperm using hysteroscopic or transrectally guided deep-horn insemination techniques.

Placement of sperm deep in the equine uterine horn allows fewer sperm to be inseminated while maintaining acceptable fertility, and has been promoted for use in circumstances when fertility would be expected to be low if standard insemination were used (e.g., semen from a subfertile stallion, or frozen-thawed semen). Two main techniques, transrectally guided (TRG) and hysteroscopic (HYS) insemination, have been developed for this purpose; however, there is some controversy regarding their comparative efficacy. This study was conducted to compare pregnancy rates when mares were inseminated by TRG or HYS, using sperm numbers approaching and under the minimum threshold, resulting in reduced fertility. When 1 × 10(6) sperm were inseminated, pregnancy rates were not different (P > 0.10) between techniques HYS (10/13, 77%) and TRG (11/15, 73%). Similarly, when 0.5 × 10(6) sperm were inseminated, pregnancy rates were not different (P > 0.10) between techniques HYS (3/15, 20%) and TRG (4/13, 31%). Combined pregnancy rates for the two treatments were 13/28 (46%) for HYS and 15/28 (54%) for TRG (P > 0.10). Pregnancy rates using a subthreshold number of sperm were not significantly affected by a deep-horn insemination technique.

Endoscopic transcervical intrauterine artificial insemination in Labrador Retriever bitches.

Besides post-thawing reduced semen quality, there are some difficulties in the execution of the endoscopic transcervical intrauterine artificial insemination (AI) with frozen-thawed semen in bitches. Therefore, the aims of the present study were to evaluate behavioral and reproductive parameters (i.e., vaginal cytology and serum progesterone level) to determine time of insemination and to investigate the particularities and difficulties of this technique in bitches using fresh semen. Ten Labrador Retriever bitches were submitted to three endoscopic transcervical intrauterine AIs (with 48 h intervals). Oestrus and ovulation period were established by behaviour evaluation, progesterone assays and vaginal cytology, enabling optimal timing for AI during oestrus. During AI, the following aspects were evaluated: cervical os catheterization difficulty, semen deposition resistance, occurrence of semen backflow, and time required to perform the AI. In this study, it was possible to catheterize the cervical os in all bitches, with different degrees of difficulty, by manipulating the equipment to allow cervical visualization and catheter introduction in the cervical canal. Serial serum progesterone assays enabled estimation of LH surge day, and thus of ovulation. The pregnancy rate was 90%, with a litter size of 5.0 ± 2.6 puppies per bitch. It was concluded that the difficulties in the execution of the endoscopic transcervical intrauterine AI technique in Labrador Retriever bitches were minimized by the equipment manipulation and practical experience.

Persistent breeding-induced endometritis after hysteroscopic insemination in the mare.

Low-dose insemination has been proposed to reduce persistent breeding-induced endometritis (PBIE) in mares with delayed uterine clearance (DUC). Others proposed that hysteroscopic insemination induces an exaggerated inflammatory response and should be avoided in DUC mares. The objectives here were to evaluate presence and severity of PBIE in normal and DUC mares after hysteroscopic insemination with fresh semen, and to determine if hysteroscopy could be used in DUC mares without inducing excessive inflammation. Reproductively normal (n = 4) and DUC (n = 5) mares received four treatments in random order: uterine body insemination (UB, 1 × 10(9) spermatozoa, 20 ml), hysteroscopic insemination (HYST, 5 × 10(6) spermatozoa, 0.5 ml), sham hysteroscopic insemination (SHAM, semen extender, 0.5 ml) and hysteroscopic infusion of seminal plasma (SP, 0.5 ml). Significantly more DUC (50%) mares than normal (14%) mares accumulated intrauterine fluid 24 h post-treatment. The difference in fluid accumulation between DUC (40%) mares and normal (7%) mares was also significant 48 h post-treatment. Fluid scores were not significantly different between treatments in normal mares. However, treatments HYST and SHAM resulted in significantly higher fluid scores 24 h but not 48 h post-treatment in DUC mares. There was no effect of treatment or mare group on the percentage and total number of neutrophils in uterine fluid 48 h post-treatment. Percentage of neutrophils was correlated with duration of hysteroscopy in normal mares, with procedures lasting ≥ 9 min associated with PBIE. There was no effect of mare group, treatment or duration of hysteroscopy on pregnancy rate. Hysteroscopy induces a transient inflammation that is not more severe than that after conventional artificial insemination, suggesting no contraindication to its use in DUC mares.

Evaluation of vaginoscopy for the diagnosis of clinical endometritis in dairy cows.

The objective of the study was to evaluate the visual assessment of vaginal discharge by vaginoscopy for the diagnosis of clinical endometritis (CE) in dairy cows. In an in vivo trial, inter- and intraobserver repeatability of vaginoscopic examination (VE) was determined and the effect of transrectal palpation and experience of the investigator evaluated. Holstein-Friesian cows (n=380) were examined by vaginoscopy between 21 and 27 d in milk by 3 investigators twice. Vaginal discharge was categorized on a 4-point classification system (0=clear mucus, 1=mucus containing flecks of pus, 2=discharge containing less than 50% pus, 3=discharge containing more than 50% pus). Cows with a vaginal discharge score (VDS) of 0 were classified as healthy, whereas cows with a VDS of 1 to 3 were classified as having CE. Vaginal discharge score on a scale from 0 to 3 has moderate intra- (Cohen's kappa coefficient, κ=0.55-0.60) and interobserver (κ=0.44) repeatability. The prevalence of CE was comparable between the 3 investigators (first VE: 42.6, 34.8, and 38.7; second VE 46.8, 36.9, and 43.7%). Transrectal palpation (relative risk=0.96-1.03) or experience of the investigator (relative risk=0.9-1.1) did not affect results of VE. In an in vitro trial, sensitivity and specificity of visual assessment were determined utilizing 33 images showing yellow and pink areas in certain percentages as a reference standard. Pus was represented by yellow areas and the mucosa, including clear mucus, by pink areas. These images were visually assessed by 30 investigators via PowerPoint presentation (experiment 1) and by 23 investigators via a simulated vaginal examination (experiment 2) utilizing the same 4-point classification system. Sensitivity was 99.6 and 96.3% and specificity was 96.7 and 90.1% in experiments 1 and 2, respectively. The results provide evidence that a visual assessment conducted by vaginoscopic examination is not perfect but can be considered a reasonable measurement of vaginal discharge and is a practical tool to distinguish healthy from diseased cows.

Techniques for the insemination of low doses of stallion sperm.

CONTENTS: In the last decade, there has been a significant increase in the quality and commercial use of frozen equine semen. The emergence of new reproductive technologies, coupled with the high prices for an insemination dose from some stallions, the increasing costs of import and export and the marketing policies of stallion agents or owners in the sport horse industry has stimulated the fractionation of doses for insemination. Consequently, the sperm number and the volume of an insemination dose are significantly reduced. To deliver lower doses of sperm in lower volumes compared to the standard dose, two techniques are used in clinical practice. Semen can be delivered hysteroscopically (HI) or by rectally guiding a flexible pipette to the tip of the desired uterine horn (RI). Both techniques have been described with good success and have triggered an incentive to further reduce the number of spermatozoa without having a negative effect on fertility. This article will review the expected success of both techniques in clinical settings and will highlight their advantages and disadvantages both for the mare and stallion. In addition, some of the implications of reducing sperm numbers on the industry will be discussed. From the available information, it is evident that lower sperm numbers deposited by RI or HI to deliver the inseminate can result in acceptable pregnancy rates with fresh or frozen semen in commercial settings. These methods of insemination could have major implications in the implementation and commercialization of new and emerging technologies in the equine industry.

Transcervical hysteroscopic sterilization using cyanoacrylate: a long-term experimental study on sheep.

AIM: We investigated the transcervical hysteroscopy application of n-butyl-2-cyanoacrylate in the uterine tube lumen of a non-rodent animals (sheep) with fallopian tube dimensions similar those in humans. METHODS: Standard hysteroscopic procedures were performed on female Texel sheep (n = 26). The right and left ostia were identified. For each ewe, a urethral catheter (5Fr) was used for the delivery of 0.5 mL of saline or an equal volume of n-butyl-2-cyanoacrylate into the uterine tube. Following the procedure, ewes were housed with males of proven fertility for 90 days (equivalent to 5.5 estrous cycles). Postmortem (dye and burst pressure) and in vivo (hysterosalpingogram) testing for tube patency were both performed 90 days and 180 days following the procedure. RESULTS: All animals receiving the saline treatment became pregnant. Gross inspection of uterine tubes following n-butyl-2-cyanoacrylate treatment revealed no visceral adhesions or fibrosis. However, postmortem testing revealed total obstruction within the fallopian tubes. This was confirmed by hysterosalpingogram, in that iodine contrast did not escape into the abdominal cavity. CONCLUSION: The cyanoacrylate promoted a reliable fallopian tube obstruction without fibrosis in an animal model exhibiting a similar tube diameter to that found in women. The technique can be evaluated for efficacy in vivo using hysterosalpingography.

Clinical and subclinical endometritis in the mare: both threats to fertility.

Endometritis, a major cause of mare infertility arising from failure to remove bacteria, spermatozoa and inflammatory exudate post-breeding, is often undiagnosed. Defects in genital anatomy, myometrial contractions, lymphatic drainage, mucociliary clearance, cervical function, plus vascular degeneration and inflamm-ageing underlie susceptibility to endometritis. Diagnosis is made through detecting uterine fluid, vaginitis, vaginal discharge, short inter-oestrous intervals, inflammatory uterine cytology and positive uterine culture. However, these signs may be absent in subclinical cases. Hypersecretion of an irritating, watery, neutrophilic exudate underlies classic, easy-to-detect streptococcal endometritis. In contrast, biofilm production, tenacious exudate and focal infection may characterize subclinical endometritis, commonly caused by Gram-negative organisms, fungi and staphylococci. Signs of subclinical endometritis include excessive oedema post-mating and a white line between endometrial folds on ultrasound. In addition, cultures of uterine biopsy tissue or of small volume uterine lavage are twice as sensitive as guarded swabs in detecting Gram-negative organisms, while uterine cytology is twice as sensitive as culture in detecting endometritis. Uterine biopsy may detect deep inflammatory and degenerative changes, such as disruption of the elastic fibres of uterine vessels (elastosis), while endoscopy reveals focal lesions invisible on ultrasound. Mares with subclinical endometritis require careful monitoring by ultrasound post-breeding. Treatments that may be added to traditional therapies, such as post-breeding uterine lavage, oxytocin and intrauterine antibiotics, include lavage 1-h before mating, carbetocin, cloprostenol, cervical dilators, systemic antibiotics, intrauterine chelators (EDTA-Tris), mucolytics (DMSO, kerosene, N-acetylcysteine), corticosteroids (prednisolone, dexamethasone) and immunomodulators (cell wall extracts of Mycobacterium phlei and Propionibacterium acnes).

Successful artificial insemination in the Asian elephant (Elephas maximus) using chilled and frozen-thawed semen.

BACKGROUND: Artificial insemination (AI) using frozen-thawed semen is well established and routinely used for breeding in various mammalian species. However, there is no report of the birth of elephant calves following AI with frozen-thawed semen. The objective of the present study was to investigate the fertilizing ability of chilled and frozen-thawed semen in the Asian elephant following artificial insemination (AI). METHODS: Semen samples were collected by from 8 bulls (age range, 12-to 42-years) by manual stimulation. Semen with high quality were either cooled to 4 degrees C or frozen in liquid nitrogen (-196 degrees C) before being used for AI. Blood samples collected from ten elephant females (age range, 12-to 52-years) were assessed for estrus cycle and elephants with normal cycling were used for AI. Artificial insemination series were conducted during 2003 to 2008; 55 and 2 AI trials were conducted using frozen-thawed and chilled semen, respectively. Pregnancy was detected using transrectal ultrasonography and serum progestagen measurement. RESULTS: One female (Khod) inseminated with chilled semen became pregnant and gave birth in 2007. The gestation length was 663 days and the sex of the elephant calf was male. One female (Sao) inseminated with frozen-thawed semen showed signs of pregnancy by increasing progestagen levels and a fetus was observed for 5 months by transrectal ultrasonography. CONCLUSION: This is the first report showing pregnancy following AI with frozen-thawed semen in the Asian elephant. Successful AI in the Asian elephant using either chilled or frozen-thawed semen is a stepping stone towards applying this technology for genetic improvement of the elephant population.