We thank ZHZ for the conception and design of the study. JW and YPZ performed and supervised the operation. YH, YBX, SL contributed to data analysis. JW and WJL prepared the draft of the video. All authors participated in the revision of the manuscript. All authors read and approved the final manuscript.

All methods described here were approved by the Ethics Committee of Union Hospital Tongji Medical College, Huazhong University of Science and Technology. We obtained the patient&#39;s consent to use his surgical footage as a video after the procedure.
1. Instruments for operation
<ol>
	<li>Ensure the availability of a surgical microscope system, microsurgical instrument, and microscopic bipolar electrocoagulation.</li>
</ol>
2. Preparation for operation
<ol>
	<li>Inclusion criteria
	<ol>
		<li>Include all patients diagnosed with azoospermia who agree to undergo surgery. Have all female partners of the patients undergo gynecologic fertility testing 
		NOTE: In the presented study, patients underwent surgery at the hospital between June 2013 and July 2020. All female patients&#39; partners&#39; gynecologic fertility test results were normal.</li>
		<li>Ensure normal serum levels of follicle-stimulating hormone and testosterone in all patients.</li>
		<li>Ensure that there are no genital malformations, hypertension, diabetes, hyperthyroidism, heart disease, pelvic trauma, abnormal psychological or mental disorders, or other serious preexisting diseases or conditions.</li>
	</ol>
	</li>
	<li>Exclusion criteria
	<ol>
		<li>Exclude patients with genital malformations, abnormal psychological or mental disorders, acute urinary tract infection or urinary tuberculosis, severe abdominal trauma or surgery, severe coagulation disorders, or other contraindications to anesthesia or laparoscopy.</li>
	</ol>
	</li>
	<li>Perform an anesthesia risk assessment on patients prior to the surgery. Ask patients who have been taking aspirin to discontinue it.</li>
	<li>Mark the skin 15 cm around the perineum with a skin preparation knife to prepare for the surgery.</li>
	<li>Administer antibiotics to the patient via intravenous drip 30 min prior to surgery. Ideally, use cefoperazone sodium (2.0 g) together with 100 mL of 0.9% sodium chloride solution.</li>
	<li>Make sure that the patient is under general or intraspinal anesthesia.</li>
</ol>
3. Procedure
<ol>
	<li>Place the patient under general anesthesia with endotracheal intubation. Then, perform standard disinfection with 1% iodophor while the patient is in the supine position.</li>
	<li>Make a 3 cm incision along the mid-scrotal fold. Then, pinch one testicle and incise the flesh membrane to expose the testicular sheath and spermatic cord.</li>
	<li>Dissect the fascia between the vas deferens and the spermatic cord vessels. Separate along the space between the vas deferens vasculature and the vasculature of the spermatic cord with a vascular clamp. Secure the vas deferens with tissue forceps and prepare it for vesicocentesis.</li>
	<li>Insert a 24 G cannula needle obliquely 15&#176; downward along the long axis of the vas deferens. Pull out the needle core after the obvious tactile breakthrough, and slide the soft trocar to the lumen.</li>
	<li>Inject 2 mL of 0.9% sodium chloride solution into the vas deferens using a 5 mL syringe connected to the 24 G cannula. If resistance is high, this confirms that there is an obstruction in the vas deferens.</li>
	<li>Take a sample of fluid from the syringe and give it to an examiner to examine it under the microscope for sperm.</li>
	<li>Advance the 3-0 polypropylene suture through the trocar. If there is resistance, it indicates obstruction. Measure the length of the suture to determine the site of obstruction.</li>
</ol>
4. Postoperative care
<ol>
	<li>Make the patient stay in the ICU for ~1-2 h postoperatively, until he fully regains consciousness.</li>
	<li>Send the patient back to the ward when he fully wakes up.</li>
	<li>Implant a catheter for 6-24 h.</li>
	<li>Lift the scrotum with underwear for 1 week.</li>
</ol>

<ol><li>Chen, X. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+features+and+therapeutic+strategies+of+obstructive+azoospermia+in+patients+treated+by+bilateral+inguinal+hernia+repair+in+childhood%5BTitle%5D)+AND+%22Asian+Journal+of+Andrology%22%5BJournal%5D)">Clinical features and therapeutic strategies of obstructive azoospermia in patients treated by bilateral inguinal hernia repair in childhood.</a> Asian Journal of Andrology. 16 (5), 745-748 (2014).</li>
<li>Sparkman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bilateral+exploration+in+inguinal+hernia+in+juvenile+patients.+Review+and+appraisal%5BTitle%5D)+AND+%22Surgery%22%5BJournal%5D)">Bilateral exploration in inguinal hernia in juvenile patients. Review and appraisal.</a> Surgery. 51, 393-406 (1962).</li>
<li>Pollak, R., Nyhus, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complications+of+groin+hernia+repair%5BTitle%5D)+AND+%22The+Surgical+Clinics+of+North+America%22%5BJournal%5D)">Complications of groin hernia repair.</a> The Surgical Clinics of North America. 63 (6), 1363-1371 (1983).</li>
<li>Sheynkin, Y. R., Hendin, B. N., Schlegel, P. N., Goldstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microsurgical+repair+of+iatrogenic+injury+to+the+vas+deferens%5BTitle%5D)+AND+%22The+Journal+of+Urology%22%5BJournal%5D)">Microsurgical repair of iatrogenic injury to the vas deferens.</a> The Journal of Urology. 159 (1), 139-141 (1998).</li>
<li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+for+Vas+Deferens+Obstruction+Following+Childhood+Herniorrhaphy%5BTitle%5D)+AND+%22Urology%22%5BJournal%5D)">Treatment for Vas Deferens Obstruction Following Childhood Herniorrhaphy.</a> Urology. 112 (1), 80-84 (2018).</li>
<li>Smith, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+significance+of+sperm+heads+and+tails+within+the+vasal+fluid+during+vasectomy+reversal%5BTitle%5D)+AND+%22Indian+Journal+of+Urology%22%5BJournal%5D)">The significance of sperm heads and tails within the vasal fluid during vasectomy reversal.</a> Indian Journal of Urology. 30 (2), 164-168 (2014).</li>
<li>Jungwirth, A., Diemer, T., Kopa, Z., Krausz, C., Tournaye, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((European+Association+of+Urology+guidelines+on+male+infertility.+2017+edition%5BTitle%5D)+AND+%22%22%5BJournal%5D)">European Association of Urology guidelines on male infertility. 2017 edition.</a> , 23-24 (2017).</li>
<li>Bromage, S. J., Falconer, D. A., Lieberman, B. A., Sangar, V., Payne, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sperm+retrieval+rates+in+subgroups+of+primary+azoospermic+males%5BTitle%5D)+AND+%22European+Urology%22%5BJournal%5D)">Sperm retrieval rates in subgroups of primary azoospermic males.</a> European Urology. 51 (2), 539-540 (2007).</li>
<li>Du, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+diagnosis+of+azoospermia+and+etiologic+classification+of+obstructive+azoospermia%3A+role+of+scrotal+and+transrectal+US%5BTitle%5D)+AND+%22Radiology%22%5BJournal%5D)">Differential diagnosis of azoospermia and etiologic classification of obstructive azoospermia: role of scrotal and transrectal US.</a> Radiology. 256 (2), 493-503 (2010).</li>
<li>Pasqualotto, F. F., Pasqualotto, E. B., Agarwal, A., Thomas Jr, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Results+of+microsurgical+anastomosis+in+men+with+seminal+tract+obstruction+due+to+inguinal+herniorrhaphy%5BTitle%5D)+AND+%22Revista+do+Hospital+das+Clinicas%22%5BJournal%5D)">Results of microsurgical anastomosis in men with seminal tract obstruction due to inguinal herniorrhaphy.</a> Revista do Hospital das Clinicas. 58 (6), 305-309 (2003).</li>
<li>Shaeer, O. K. Z., Shaeer, K. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laparoscopy-assisted+pelvi-scrotal+vasovasostomy%5BTitle%5D)+AND+%22Andrologia%22%5BJournal%5D)">Laparoscopy-assisted pelvi-scrotal vasovasostomy.</a> Andrologia. 36 (5), 311-314 (2004).</li>
</ol>

The authors have no conflict of interest and nothing to disclose.

As mentioned before, the location of the vas deferens stump cannot be determined at the inguinal incision, which requires exploration of the original inguinal incision. In this study, we can use this simple operation to determine the site of obstruction and make surgical decisions. If the site of obstruction is in the original incision, MVV can be considered. If the vas deferens injury is not found in the inguinal canal, we can use lapa-VV or SV.
When pasty fluid without sperm was found and the obstruction of the vas deferens was 2 cm higher than the original incision, the procedure was stopped and the incision was sutured. However, because of the long operation time, costly procedure, large surgical incision, and uncertain results of the combined lapa-VV-MVE operation, many patients might refuse to undergo this operation and opt for ART6.
Patients in whom natural pregnancy could not be achieved, mainly because of poor semen quality, considered intracytoplasmic sperm injection (ICSI) or artificial insemination as another option. Artificial insemination by the male or ICSI can be attempted more than once. Complications were recorded after 12 months7,8.&#160;Patients with long-term epididymal stasis may develop epididymal obstruction causing pasty vasal fluid without sperm9. The combination of MVV and MVE may be a potential therapeutic option in some patients10.
Consistent with open surgery, VV is a difficult inguinal operation after herniorrhaphy, and the site of obstruction cannot be identified preoperatively. Overexploration may result in injury to the testicular and vas deferens vessels, or the recurrence of hernia repair11. Surgery is difficult to perform, especially when the remnants of the vas deferens cannot be found. Therefore, intraoperative evaluation of the site of obstruction is very important.
In our study, there are several possibilities for exploration: if a smear reveals that there is no sperm in the cannula fluid, there may be a concomitant obstruction at the junction of the vas deferens and epididymis. If the obstruction of the vas deferens is 2 cm higher than the top of the original incision palpated by the polypropylene suture, consideration may be given to undergoing ART directly. If the obstruction site is in the original incision, MVE + MVV may be considered. If a smear demonstrates that sperm is present in the fluid of the cannula while the obstruction site with the 3-0 polypropylene suture is in the incision of the original hernia repair, the original inguinal incision can be examined directly. If a smear demonstrates that sperm is present in the cannula fluid while the 3-0 polypropylene suture is within 2 cm of the top of the original incision for evidence of obstruction, laparoscopy-assisted vas deferens exploration can be considered, and the proximal vas deferens can be pulled out of the external inguinal ring for anastomosis. If a smear demonstrates that sperm is present in the fluid of the cannula while the 3-0 polypropylene suture is more than 5 cm from the upper edge of the original incision, seminal vesicle dysplasia or ejaculatory duct obstruction may be present. In combination with preoperative semen volume and seminal vesicle magnetic resonance imaging (MRI), endoscopic examination of the seminal vesicle could be considered.
Simple surgery with a 24 G cannula and a 3-0 polypropylene suture results in little injury to the vas deferens, optimizes the surgical process, increases the intensity of surgical decision making, and provides a simple and effective method to maximize the patient&#39;s interests.

Microsurgical vasovasostomy (MVV) is a common treatment for vas deferens obstruction after herniorrhaphy in childhood1,2. The site of obstruction cannot be identified preoperatively, and it is not clear whether sperm are present in the vas deferens or not. This may lead to difficulty in selecting the incision and injury to the vas deferens during semi-open exploration, which decreases the success rate of anastomosis, prolongs the operation time, and increases the risk of abdominal exploration3,4. 
Injury to the vas deferens in the inguinal canal is likely an after-effect of inguinal hernia surgery in childhood, which can usually be caused by ligation, incision, or overstretching1. The rate of azoospermia after herniorrhaphy in childhood is approximately 0.8%-2.0% in pediatric patients. However, in the infertile population, the rate of iatrogenic obstruction is significantly increased, with a history of vas deferens injury in 7.2% of andrological procedures2,3. Surgical exploration of the inguinal region is usually performed to look for vas deferens remnants for anastomosis4. In previous cases, the ruptured end of the vas deferens is usually not found during inguinal exploration, which leads to the termination of the operation and increases surgical trauma to the patient. Therefore, it is very important to use a simple method to determine the location of the obstruction to clarify the following surgical procedures. 
In this study, a 24 G cannula was used to puncture the vas deferens to confirm the presence of sperm in it, and a 3-0 polypropylene suture was passed through the cannula to determine the location of the obstructed site. Based on this simple operation, we can determine the next surgical procedure. After practicing and summarizing the clinical operations, our specific surgical method is presented below.

<table><tbody><tr><td>0.9% sodium chloride solution</td><td>Guangdong Otsuka Pharmaceutical Co. LTD</td><td>&amp;nbsp;21M1204&amp;nbsp;</td><td>Inject into the vas deferens</td></tr><tr><td>1% iodophor</td><td>Guangzhou Qingfeng Disinfection Products Co., LTD&amp;nbsp;</td><td>Q/QFXD2</td><td>Skin disinfection</td></tr><tr><td>24G cannula needle</td><td>Suzhou Linhua Medical Instrument Co., LTD</td><td>20193141896</td><td>Size:24G, Type:Pen type, with wings, with injection port, Y type</td></tr><tr><td>3-0 monofilament polypropylene</td><td>Covidien Medical Equipment international trading (Shanghai) Co., LTD</td><td>DOL2861y</td><td>Taper Point, Size 3-0, Blue, 36&quot;, Needle V-20, 1/2 Circle</td></tr><tr><td>5 mL syringe</td><td>&amp;nbsp;Kindly Medical, Shanghai</td><td>&amp;nbsp;K20210826</td><td>Inject 0.9% sodium chloride solution into the vas deferens</td></tr><tr><td>F16 Catheter</td><td>&amp;nbsp;Guangzhou Weili Co., Ltd&amp;nbsp;</td><td>20190612</td><td>Drainage of urine</td></tr><tr><td>micro haemostatic forceps</td><td>Shanghai Medical Instrument (Group) Co., LTD.</td><td>W40350</td><td>Used in surgical procedures</td></tr><tr><td>micro scissors</td><td>Shanghai Medical Instrument (Group) Co., LTD.</td><td>WA1010</td><td>Used in surgical procedures</td></tr><tr><td>micro tweezers</td><td>Shanghai Medical Instrument (Group) Co., LTD.</td><td>WA1020</td><td>Used in surgical procedures</td></tr><tr><td>Olympus microscope</td><td>Olympus Corporation</td><td>Scipu001158</td><td>4&amp;times;N.A.0.10 W.D.22.0mm&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 10&amp;times;N.A.0.25 W.D.10.5mm&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 40&amp;times;N.A.0.65 W.D.0.56mm&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 100&amp;times;N.A.1.25 W.D.0.13mm&amp;nbsp;</td></tr><tr><td>Operating microscope system</td><td>&amp;nbsp;Carl Zeiss Co., Ltd&amp;nbsp;</td><td>OPMI VARIO 700</td><td></td></tr></tbody></table>

clinical application of 24 g cannula needle and 3-0 polypropylene suture in vas deferens exploration

The goal of this article is to present the application of a 24 G cannula and 3-0 polypropylene suture as a simple method for the exploration of the vas deferens. During the exploration of the vas deferens, a 24 G cannula needle was used to puncture it. The fluid in the smear confirmed the presence of sperm, to determine whether or not there was concomitant obstruction at the junction of the epididymis and the vas deferens. Then, a 3-0 polypropylene suture (this suture specification has the advantages of a smooth surface, robust quality, and can be passed through a 24 G cannula needle) was passed through the cannula needle to probe the location of the obstructed site. With this technique, exploration of the vas deferens could be more targeted and accurate.

There were 67 patients enrolled in the study. As shown in Table 1, the mean age of the patients was 28.8 &#177; 3.7 years (range: 23-45 years). The time since herniorrhaphy was 24.5 &#177; 3.2 years (range: 21-43 years). The female partners were 25.2 &#177; 3.2 years (range: 23-42 years) years old. A total of 11 (16.4%) patients had pain in the scrotum. Four patients (6.0%) were underweight (BMI &#60; 18.5 kg/m2), 53 (79.1%) had normal weight (BMI 18.5-24.9 kg/m2), eight (11.9%) were overweight (BMI 25-29.9 kg/m2), and two (3.0%) were obese (BMI &#62; 30 kg/m2) .
In our study, the sites of obstruction were classified into three groups. (1) The obstruction of the vas deferens is at the incision of the original hernia repair (&#62;2 cm); (2) the obstruction of the vas deferens is 2 cm higher than the original incision; (3) the obstruction of the vas deferens is more than 5 cm from the upper edge of the original incision. Table 2 shows the location of vas deferens obstruction and the results according to the surgical method in the different subgroups. The smear showed that 25 cases (37.3%) had no sperm in the cannula fluid. Of these, three (12.0%) underwent bilateral VV surgery with the obstruction site in the original incision, and the patency rate was 33.3% (one of three). In total, 22 (88.0%) underwent assisted reproductive technology (ART), in which the obstruction of the vas deferens was 2 cm higher than the upper edge of the original incision. The smear detected sperm in the fluid of the cannula needle in 42 (62.7%) cases. Of these, 29 (69.0%) underwent bilateral VV surgery with the obstruction site in the original incision, and the patency rate was 79.3% (23 of 29). A total of 10 (23.0%) underwent laparoscopy-assisted (lapa)-VV, with the obstruction site 2 cm higher than the original incision, and the patency rate was 40% (four of 10). A further three (7.10%) underwent seminal vesiculoscopy (SV), in which the obstruction site was 5 cm higher than the original incision, and the patency rates was 33.3% (one of three).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63833/63833fig01.jpg" /> 
Figure 1: Schematic of the procedure. (A) The thickened vas deferens was separated. (B) A 3-0 polypropylene suture was passed through the cannula needle to probe the position of the obstructed site. (C) The vas deferens was identified beneath the peritoneum. (D) The vas deferens was mobilized with its blood supply off scar tissue. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63833/63833fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Characteristics</td>
			<td>N</td>
		</tr>
		<tr>
			<td>Patient age, years</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Mean&#177;SD&#160;</td>
			<td>28.8&#177;3.7</td>
		</tr>
		<tr>
			<td>&#160; Range&#160;</td>
			<td>23-45</td>
		</tr>
		<tr>
			<td>Time since herniorrhaphy, years</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Mean&#177;SD&#160;</td>
			<td>24.5&#177;3.2</td>
		</tr>
		<tr>
			<td>&#160; Range&#160;</td>
			<td>21-43</td>
		</tr>
		<tr>
			<td>Female partner age, years</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Mean&#177;SD&#160;</td>
			<td>25.2&#177;3.2</td>
		</tr>
		<tr>
			<td>&#160; Range&#160;</td>
			<td>23-42</td>
		</tr>
		<tr>
			<td>With scrotal pain, n (%)</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Yes&#160;</td>
			<td>11(16.4%)</td>
		</tr>
		<tr>
			<td>&#160; No</td>
			<td>56(83.6%)</td>
		</tr>
		<tr>
			<td>BMI</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Underweight (&#60;18.5 kg/m2)</td>
			<td>4(6.0%)</td>
		</tr>
		<tr>
			<td>&#160; Normal (18.5-24.9 kg/m2)</td>
			<td>53(79.1%)</td>
		</tr>
		<tr>
			<td>&#160; Overweight (25-29.9 kg/m2)</td>
			<td>8(11.9%)</td>
		</tr>
		<tr>
			<td>&#160; Obese (&#8805;30 kg/m2)</td>
			<td>2(3.0%)</td>
		</tr>
		<tr>
			<td>BMI, body mass index.&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Basic information of the patients. Abbreviation: BMI = body mass index.&#160;Data for this table is reprinted with permission from Wang et al.5
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sperm exist in the vas deferens</td>
			<td>Patients, n (%)</td>
			<td>The site of obstruction</td>
			<td>Patients, n (%)</td>
			<td>Treatment (%)</td>
			<td>Patency n (%)</td>
		</tr>
		<tr>
			<td rowspan="2">No</td>
			<td rowspan="2">25(37.3)</td>
			<td>&#60; 2cm</td>
			<td>3(12.0)</td>
			<td>MVE+MVV</td>
			<td>1(33.3)</td>
		</tr>
		<tr>
			<td>&#62;2cm</td>
			<td>22(88.0)</td>
			<td>ART</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Yes</td>
			<td>42(62.7)</td>
			<td>&#60;2cm</td>
			<td>29(69.0)</td>
			<td>Bi-VV&#160;</td>
			<td>23(79.3)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>&#62;2cm</td>
			<td>10(23.8)</td>
			<td>Lapa-VV&#160;</td>
			<td>4(40.0)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>&#62;5cm</td>
			<td>3(7.1)</td>
			<td>SV&#160;</td>
			<td>1(33.3)</td>
		</tr>
	</tbody>
</table>
Table 2: The site of vas deferens obstruction and the results based on operative manners in different subgroups. Abbreviations: MVE = microsurgical vasoepididymostomy; MVV = microsurgical vasovasostomy; Bi-VV = bilateral vasovasostomy; lapa-VV = laparoscopy-assisted vasovasostomy; SV = seminal vesiculoscopy; ART = assisted reproductive technology.

Watch this Scientific Journal Video about Clinical Application of 24 G Cannula Needle and 3-0 Polypropylene Suture in Vas Deferens Exploration at JoVE.com

Clinical Application of 24 G Cannula Needle and 3-0 Polypropylene Suture in Vas Deferens Exploration

This protocol presents the clinical application of a 24 G cannula and 3-0 polypropylene suture as a simple and effective method for the exploration of the vas deferens.

The goal of this article is to present the application of a 24 G cannula and 3-0 polypropylene suture as a simple method for the exploration of the vas ...

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2.6K Views.  Huazhong University of Science and Technology. This protocol presents the clinical application of a 24 G cannula and 3-0 polypropylene suture as a simple and effective method for the exploration of the vas deferens.

Video: Clinical Application of 24 G Cannula Needle and 3-0 Polypropylene Suture in Vas Deferens Exploration

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The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of G&ouml;ttingen minipigs in biomedical research.

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Biology

Application of Straight-needle, Three-tailed, Knot-free, Peritoneal Sutures in Laparoscopic Transabdominal Preperitoneal Hernia Repair

Laparoscopic transabdominal preperitoneal hernia repair (TAPP) is one of the most widely used methods in inguinal hernia surgery. After the mesh is placed, the peritoneum must be resutured to avoid contact with the tissues and organs in the abdominal cavity. If the peritoneal suture time is too long, the operation and anesthesia time will be prolonged, increasing the burden on the patient. Moreover, improper suture methods cause serious consequences, such as intestinal obstruction and mesh infection.
The straight-needle suture method transforms the three-dimensional spatial configuration of the needle holder and the arc needle tip into a two-dimensional planar structure, which greatly reduces the difficulty of suturing. The three-tailed knot can be anchored at the beginning of the suture by its friction and button effect, which has an exact fixation effect. Thus, the suture does not easily slip, and the time to complete the suturing is shortened. Compared with the traditional suture method, the operator can suture the peritoneum more quickly, beginners can pass through the difficult learning curve faster, and skilled operators can also shorten the total operation time of TAPP to a certain extent. Thus, this suture method is extremely amenable to clinical application.

Here, we present a protocol to introduce a new peritoneal suture method. This method is called straight-needle, three-tailed, knot-free suture, and we will outline the manufacturing method and clinical application of this suture in detail.

Laparoscopic transabdominal preperitoneal hernia repair (TAPP) is one of the most widely used methods in inguinal hernia surgery. After the mesh is ...

Vessel-Sparing Microsurgical Longitudinal Intussusception Vasoepididymostomy to Treat Epididymal Obstructive Azoospermia

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of epididymal OA since 2000. There are two challenges in classic microscopic vasoepididymostomy. First, anastomosis of the vas deferens and epididymis is performed with double-needle sutures. However, there is a lack of good-quality and cost-effective double-needle sutures in China, which leads to increased difficulty and poor success rates of anastomosis. Second, the separation of the vas deferens does not retain vasculature, although the vas deferens vasculature plays an important role in the blood supply to the vas deferens, epididymis, and testis. This affects the blood supply to the anastomotic area and epididymis.
Therefore, this team has made innovative improvements to address these problems. Good-quality, cost-effective, single-needle sutures, which are easy to purchase in China and other countries, were used in microsurgical longitudinal intussusception vasoepididymostomy. This can optimize the operation procedure and shorten the operation time while ensuring the success rate of the anastomosis. The surgical method of preserving the vas deferens vessels was innovatively proposed because the etiology of epididymal OA is mostly inflammatory in China. The protection of the blood supply to the vas deferens and epididymis is maximized using microsurgical forceps to separate and protect the vasculature. Patency reached 81.7% in the postoperative follow-up, indicating a better surgical treatment effect.

The present protocol describes a vessel-sparing, longitudinal intussusception vasoepididymostomy using readily available single-needle sutures in China, a safe and effective procedure, which may improve patients' patency and natural pregnancy rates.

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of ...

A Modified Vessel-Sparing Microsurgical Vasoepididymostomy

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, preserving the vasal vessels during MVE might better simulate the normal physiological structure and be meaningful for patients who have undergone varicocelectomy. Nevertheless, preserving the vasal vessels might elevate the risk of increasing the tension of anastomosis, affecting the patency rate and leading to delayed postoperative bleeding. Therefore, we developed a novel vessel-sparing MVE to make it safer. Here is a summary of the improvements to the procedure. 1) The retrograde dissociation of the vasal vessels on the proximal testicular side was adopted as the main method, and the anterograde dissociation of the vasal vessels on the distal testicular side was adopted as a supplement to dissociate the vasal vessels to be preserved. This improvement ensures the blood supply to the vas deferens that will be used for anastomosis and also provides longer vasal vessels, which reduces the tension of anastomosis. 2) By fixing the vas deferens to be anastomosed and the broken end of the vas deferens, the free vasal vessels get fixed, which resolves the problem of transmission of vas tension to the vasal vessels and reduces the risk of vasal vessel hemorrhage. 3) Dissociation of the vas deferens after opening the tunica vaginalis increases the mobilization of the vas deferens, which also makes the new procedure easier to complete. The evaluation of the outcomes of this new procedure showed that no significant postoperative complications occurred in the patients, and the patency rate was no different from that of the conventional procedure. Therefore, this new, improved procedure can be considered safe, with satisfactory postoperative results.

Here, we present a protocol to preserve the vasal vessels in microsurgical vasoepididymostomy. The surgical security is enhanced by preserving the vasal vessels using a retrograde-anterograde dissociation and fixing the vasal vessels.

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

Combination of High Ligation and Intraoperative Embolization using Polidocanol for Treatment of Varicoceles

Varicoceles are dilated veins within the pampiniform plexus and are relatively common in the general male population. The spermatic vein has many branches in the scrotal segment and then gradually merges into 1-2 trunks after passing through the internal inguinal ring. The key to a successful varicocelectomy is to ligate all the spermatic veins while protecting the testicular arteries and spermatic lymphatic vessels from damage. The small veins, including the branches of spermatic veins and collateral veins, are easily missed for ligation during conventional high ligation of varicocele, which has been suggested as a major cause of postoperative recurrence. Although microsurgery effectively reduces the risk of missing ligation of the spermatic veins during surgery, it has several shortcomings, such as long operation time and a steep learning curve. More importantly, this technique is difficult to carry out in primary hospitals due to the requirement of specialized equipment. Therefore, an attempt to modify the traditional high ligation aiming to reduce the postoperative recurrence rate has been carried out here. The protocol here combines traditional high ligation with intraoperative embolization to seal off the branches of the spermatic vein and collateral veins. We rapidly injected foamed sclerosant into the internal spermatic vein under direct observation after separation of the spermatic vein and then ligated all the veins. The foamed sclerosant through the varicose vein hampers endothelial cell growth, promotes the growth of thrombus and fibrosis, and ultimately forms fibrous streaks that permanently fill the venous. The results showed a more satisfactory effect on reducing the postoperative recurrence rate compared with traditional high ligation. Since this protocol is simple to carry out and has better results in reducing the recurrence rate, this can be an alternative surgical method for the treatment of varicocele, especially in primary hospitals.

To reduce the postoperative recurrence rate of varicocelectomy, we combined high ligation of varicocele with intraoperative embolization. We injected polidocanol from the spermatic vein during surgery to embolize the branches of the spermatic vein and collateral veins. This is an alternative surgical method for the treatment of varicocele.

Varicoceles are dilated veins within the pampiniform plexus and are relatively common in the general male population. The spermatic vein has many branches ...

Grade III Varicocele Surgical Treatment using Spermatic Vein-Superficial Abdominal Vein Shunt

Microsurgical varicocelectomy is the most commonly used method for the treatment of varicocele (VC) in recent years. However, it is technically demanding with the risk of damaging the normal anatomical structure of the spermatic cord, such as the cremaster muscle, testicular artery, and vas deferens during the pampiniform plexus ligation. Also, traditional varicocelectomy hinders the drainage of the stagnant venous blood of the affected testicle, resulting in a persistent scrotal appearance of varicose veins and slower remission of swelling sensation in postoperative patients with grade III VC. Therefore, we developed a retroperitoneal varicocelectomy with a microscopical spermatic venous-superficial vein of the abdominal wall bypass procedure. The spermatic vein was transected and ligated proximally through the retroperitoneal space. Then, the distal spermatic vein was freed and passed through the internal ring; under the skin of the groin, a microscopic vascular anastomosis was performed to build the bypass of the distal spermatic vein and proximal inferior epigastric vein. The high ligation facilitates the protection of the normal anatomy of the spermatic cord, and the venous bypass allows rapid testicular blood drainage, which can effectively improve the degree of varicocele, testicular pain, and even spermatogenic function. In conclusion, the present protocol describes a promising way to reconstruct the spermatic return through high retroperitoneal ligation of the spermatic vein and anastomosis of the spermatic vein-inferior epigastric vein, which resulted in faster and more obvious improvement in symptoms and better prognosis of grade III VC.

Here, we present a protocol for grade III varicocele surgical treatment that aims to rebuild the venous drainage of the spermatic cord by a spermatic vein-superficial abdominal vein shunt performed under the microscope.

Microsurgical varicocelectomy is the most commonly used method for the treatment of varicocele (VC) in recent years. However, it is technically demanding ...

Bilateral Spermatic Nerve Blocks and Vasectomy Procedures in Males

Optimizing the Modified No-Scalpel Vasectomy Technique

No-scalpel vasectomy (NSV) is a commonly used contraceptive method for males. In previous applications of this surgical method, issues such as knot detachment and postoperative recanalization resulted in a reduced contraceptive success rate after vasectomy. This new technique innovatively modifies the ligation procedure during NSV to address knot detachment and postoperative recanalization. Two ligations of the testicular end of the vas deferens and one ligation of the abdominal end are completed before transection of the vas deferens. A fourth ligation of the abdominal end is performed using the suture tails of the third ligature of the testicular end after transection of the vas deferens. The fifth ligation of the abdominal end is performed using the suture tails of the first ligature of the testicular end. These modifications strengthen the ligations and reduce the postoperative recanalization rate. Additionally, the two stumps are gathered together, creating better conditions for a possible vasovasostomy in the future, thereby achieving overall better outcomes with vasectomy.

A modified no-scalpel vasectomy technique reduces the postoperative recanalization rate. This protocol describes a modified no-scalpel vasectomy technique that can reduce the postoperative recanalization rate.

No-scalpel vasectomy (NSV) is a commonly used contraceptive method for males. In previous applications of this surgical method, issues such as knot ...

Intraduodenal Cannulation in a Rat Model for Immunopathology Studies

Source: Trevaskis, N. L. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52389">The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates intraduodenal cannulation in a rat model. After anesthetizing the rat, the duodenum is located and a small puncture is created. A J-shaped cannula with a beveled tip is carefully inserted and secured, and the incision is closed. The cannulated rat model enables intra-intestinal infusion for immunopathology studies.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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