A Study on In Vitro Fertilization and Embryo Transfer in Rabbits

Pang, Myung Geol,Chung, Yung Chai,Kim, Chang Keun 中央大學校 遺傳工學硏究所 1988 遺傳工學硏究論集 Vol.1 No.1

수토끼의 개체별 사출정자의 체외수정능 획득과 체외수정능력의 차이를 비교하고, 체외수정란의 체외발생능력을 조사함과 동시에 이식수정란의 분만율을 조사하기 위해 실험이 수행되었다. 사출정자의 체외수정능 획득은 HIS처리후 18시간 DM에서 전배양하였고, 체외수정은 과배란 처리후 회수된 난자와 38℃ CO₂배양기내에서 36시간 동안 배양하였다. 체외수정란은 m F-12에서 배반포시기까지 발달을 조사하였고, 일부는 이식하여 분만율을 조사하였다. 1. 18시간 전배양한 체외수정능 획득정자의 체외수정률(난할율)은 수토끼 개체간에 차이가 컸으며, 그범위는 0-61.1%였다. 2. 체외수정란의 체외발생능력을 조사한 결과 150개의 수정란중 70개(46.4%)가 배반포시기까지 발생하였다. 3. 체외수정란을 난관(4세포기 30개)과 자궁(상실배 29개)에 각각 3수의 수란토에 이식하여 6두(20.0%)와 3두(10.3%)의 자토를 분만하였으며, 6수의 수란토중 3수(5%)가 정상분만을 하였다.

Fluorescence in Situ Hybridization 시행을 위한 인간정자 탈응축법의 적정화

방명걸,Pang, Myung-Geol 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.3

Studies were conducted to determine the efficiency of decondensation protocols. Sperm obtained from seven normal donors was immediately washed after liquefaction and then decondensed using the method of West et al. (1989) and my original protocol. My optimized protocol entailed mixing 1 ml aliquots of semen with 4 ml phosphate buffered saline (PBS). Following centrifugation, pellets were resuspended in 1 ml PBS containing 6 mM EDTA. After centrifugation, pellets were resuspended in 1 ml PBS containing 2 mM dithiothreitol at $37^{\circ}C$ for 45 min. Following mixing with 2 ml PBS and centrifugation, pellets were resuspended by vortexing. While vortexing, 5 ml of fixative were gently added. Slide preparation was accomplished using the smear method and it was stored at $4^{\circ}C$. When comparing these protocols, the degree of sperm decondensation and head swelling was monitored by measuring nuclear length, area, perimeter, and degree of roundness using FISH analysis software. Apparent copy number for chromosome 1 and, separately, for the sex chromosomes was determined by FISH using satellite DNA probes for loci DIZ1, DXZ1 and DYZ3. Sperm treated by my decondensation protocol showed significant increases (p<0.05) in length, area, perimeter, and degree of roundness. There was a significant decrease (p<0.05) in the frequency of nuclei displaying no signal but no change in the frequency of nuclei with two signals in samples decondensed by my protocol. My data suggested that decondensation using my original protocol may lower the frequency of cells with spurious "nullisomy" due to hybridization failure without inducing spurious "disomy" resulting from increased distances between split signals.

Porcine seminal protein-I and II mRNA expression in boar spermatozoa is significantly correlated with fertility

Kang, Saehan,Pang, Won-Ki,Ryu, Do-Yeal,Song, Won-Hee,Rahman, Md Saidur,Park, Yoo-Jin,Pang, Myung-Geol Elsevier 2019 Theriogenology Vol.138 No.-

<P><B>Abstract</B></P> <P>In recent years, genomic and proteomic biomarkers have been identified for the diagnosis of male fertility to overcome the limitations of conventional semen analysis. Owing to the limited genes available so far, the single gene approach is commonly adopted for analyzing the phenotype of interest. However, the single-gene approach is less effective than multiple-gene strategies for diagnosing a desirable phenotype. Herein, we investigate the ability of two fertility-related genomic markers (porcine seminal protein (PSP)-I and PSP-II) in spermatozoa to predict boar litter size in addition to conventional semen parameters. First, we examined different semen parameters (motility, motion kinematics, and capacitation status) and gene expression in high- and low-litter size boar spermatozoa. Then, we evaluated the correlation of these parameters with the fertility of 21 Yorkshire boars. Finally, we investigated the efficacy of single/combined markers to predict male fertility using a comprehensive statistical model. Our result showed that there were no significant differences in sperm motility, motion kinematics, or capacitation status, however, the mRNA expression of PSP-I and PSP-II in spermatozoa was significantly different in high- and low-litter size boars. In the individual screening test, the expression of both genes was negatively correlated with boar fertility (r = 0-0.578 and −0.456, respectively), whereas only hyperactivation (HYP) showed a positive correlation (r = 0.444) among the tested semen parameters. As single markers, PSP-I and PSP-II have a better diagnostic power to predict boar fertility, regardless of HYP, in quality assessment analyses. In addition, when these markers were combined, the positive predictive value, negative predictive value, and overall test effectiveness for fertility detection were improved. Surprisingly, when PSP-I and PSP-II were considered together, the deviation of the predicted average litter size between high- and low-litter size boars was 1.77. Based on the findings, we suggest that the use of genomic markers in spermatozoa rather than commonly analyzed semen parameters may be more accurate for evaluating male fertility. Moreover, using a combination of markers could increase the overall accuracy of (in)fertility predictions, and thus, could be considered for field application.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PSP-I and PSP-II expression in spermatozoa was negatively correlated with boar fertility. </LI> <LI> The combined use of PSP-I and PSP-II showed improved overall test accuracy for fertility detection. </LI> <LI> The combined use of PSP-I and PSP-II displayed maximum piglets increased. </LI> </UL> </P>

Over Expression of Aminopeptidase N has Negative Effect on Boar Fertility

Won-Ki Pang,Kyu-Ho Kang,Ki-Uk Kim,Ki-Jin Kwon,Sae-Han Kang,Amena Khatun,Do-Yeal Ryu,Md Saidur Rahman,Myung-Geol Pang 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

Artificial insemination is a commonly used technology in the porcine industry that directly linked to the genetic upgradation of future progeny and profitable farm management. Therefore, accurate prediction of boar fertility is a matter of paramount importance. Although prediction of semen/sperm fertility using conventional analysis provides a preliminary assumption of male fertility, its practical value is limited. Protein content in the cell provides an important and fascinating insight to the dynamics of cell function. Therefore, the study of protein function in spermatozoa and their corresponding relationship with male fertility might be an excellent alternative tool for more accurate prediction of male fertility. Recently we demonstrated that aminopeptidase N (APN) activity is negatively correlated with motility and fertility of mice spermatozoa. Here, we investigated whether APN is also correlated with the fertility of boar. Spermatozoa were collected from high and low fertility boar based on their field fertility data (average litter size 13.2±0.06 and 11.1±0.14, respectively). Simultaneously, APN levels were examined by Western blotting using the corresponding antibody. In addition, spermatozoa from both fertility groups were examined by conventional semen analysis such as computer assisted sperm analysis (CASA) and chlorotetracycline (CTC) staining. Our data showed a significant higher APN expression in low fertility boar spermatozoa compare to the fertile counterpart (p<0.05). Interestingly, no significant difference was noticed in motility, motion kinematics, and capacitation status between both groups. Based on this findings, it is tempting to speculate that APN activity might directly correlate with boar fertility. Therefore, APN activity in spermatozoa is more accurate and sensitive biomarker for the detection of boar fertility then conventional semen analysis. However, further studies and needed to confirm our initial findings

The Effects of Isotypes and Regional Distribution of Antisperm Antibodies on Semen Parameters and Fertilizing Ability

방명걸,문신용,Pang, Myung-Geol,Moon, Shin-Yong The Korean Society for Reproductive Medicine 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.1

항정자항체의 종류 및 존재부위가 정액성상 및 수정능력에 미치는 영향을 조사하였다. 항정자항체의 종류 및 존재부위는 immunobead binding test에 의하여 시행하였으며, 정자와 수정능력은 투명대제거 햄스터 난자 침입법에 의하여 시행하였다. 항정자항체는 정자수, 운동성 및 운동지수에 악영향을 끼쳤으며, 수정능력에도 악영향을 끼쳤다. 항정자항체의 존재부위에 따른 차이는 보이지 않았다. 항정자항체 IgG가 정자두부 혹은 정자미부에 존재할 경우 및 항정자항체 IgA가 정자미부에 존재할 경우 수정능력을 크게 감소시켰다. To investigate the influences on semen parameters and fertilizing capacity of immunoglobulin (Ig) isotypes and regional distribution of antisperm antibody (ASA) on the human sperm surface. Sixty-seven ASA-positive patients were compared with 96 ASA-negative donors. ASAs in semen showed significant negative effects on both semen parameters and fertilizing capacity; in those with ASAs in the sperm head and/or tail, the reductions were significant. In the head as well as the tail, there was close correlation between fertilizing capacity and both IgG and IgA. Both semen parameters and fertilizing capacity are significantly affected by the presence of ASA in semen. In particular, antibodies IgG to sperm head and/or tail, and antibodies IgA to sperm tail appeared to have a highly detrimental effect on fertilizing capacity.

The effect of male circumcision on sexuality

Kim, DaiSik,Pang, Myung-Geol Blackwell Publishing Ltd 2007 BJU International Vol.99 No.3

<P>OBJECTIVE</P><P>To prospectively study, using a questionnaire, the sexuality of men circumcised as adults compared to uncircumcised men, and to compare their sex lives before and after circumcision.</P><P>SUBJECTS AND METHODS</P><P>The study included 373 sexually active men, of whom 255 were circumcised and 118 were not. Of the 255 circumcised men, 138 had been sexually active before circumcision, and all were circumcised at >20 years of age. As the Brief Male Sexual Function Inventory does not specifically address the quality of sex life, questions were added to compare sexual and masturbatory pleasure before and after circumcision.</P><P>RESULTS</P><P>There were no significant differences in sexual drive, erection, ejaculation, and ejaculation latency time between circumcised and uncircumcised men. Masturbatory pleasure decreased after circumcision in 48% of the respondents, while 8% reported increased pleasure. Masturbatory difficulty increased after circumcision in 63% of the respondents but was easier in 37%. About 6% answered that their sex lives improved, while 20% reported a worse sex life after circumcision.</P><P>CONCLUSION</P><P>There was a decrease in masturbatory pleasure and sexual enjoyment after circumcision, indicating that adult circumcision adversely affects sexual function in many men, possibly because of complications of the surgery and a loss of nerve endings.</P>

Porcine seminal protein-I and II in boar spermatozoa are candidate biomarkers for prediction of litter size

Saehan Kang,Won-Ki Pang,Do-Yeal Ryu,Won-Hee Song,Md Saidur Rahman,Myung-Geol Pang 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

The ability of conventional semen analysis to predict male fertility is questionable. Since the prediction of male fertility is extremely of importance for the artificial insemination and profitable farm managements in animals, the development of highly sensitive biomarker of male fertility is a prime concern. Porcine Seminal Protein I (PSP-I) and Porcine Seminal Protein II (PSP-II) have been known that they are related with motility, and viability of spermatozoa. Thus, we investigated PSP-I and PSP-II level in boar spermatozoa to predict boar’s fertility. The expressions of PSP-I and PSP-II in spermatozoa from 21 individual boars with different fertility and litter size (litter size ranges from 10.3 – 14.2) were examined using qRT-PCR. Litter size was determined in 530 saws after artificial insemination (AI). In addition, sperm motility, motion kinematics, and capacitation status were measured using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining, respectively. PSP-I and PSP-II showed significantly negative correlation with litter size (r=0.578; P=0.006 and r=0.456; P=0.038, respectively). Furthermore, receiver-operating curves (ROC) was used to determine the accuracy for the prediction of boar fertility. Therefore we divided into 2 groups based on the median value of litter size. When selecting higher litter size group, PSP-I can predict litter size with overall accuracy 90.48% (sensitivity 88.89, specificity 91.67, negative predictive value 91.67, and positive predictive value 88.89) and PSP-II can predict with overall accuracy 81.82% (sensitivity 55.56, specificity 100.00, negative predictive value 76.47, and positive predictive value 100.00). Interestingly, PSP-I and PSP-II were found to increase 0.76 pups than average litter size (average 12.48) in tested boars. To best of our knowledge, this study is the first trial to investigate the correlation between PSP-I, PSP-II, and litter size. Therefore, we suggest that PSP-I and PSP-II could be considered as promising biomarkers for predicting male fertility and litter size outcome in field condition.

Sperm solute carrier family 9 regulator 1 is correlated with boar fertility

Kim, Ki-Uk,Pang, Won-Ki,Kang, Saehan,Ryu, Do-Yeal,Song, Won-Hee,Rahman, Md Saidur,Kwon, Woo-Sung,Pang, Myung-Geol Elsevier 2019 Theriogenology Vol.126 No.-

<P><B>Abstract</B></P> <P>Predicting male fertility is extremely important for artificial insemination and profitable farm management. Conventional semen assessment together with computer-assisted sperm analysis is widely used to predict male fertility under field conditions. However, the clinical validation and sensitivity of these methods remain unclear. Therefore, a new approach is needed to predict male fertility. Here, we investigated the use of a transcriptomic marker (solute carrier family 9, subfamily A, member 3, regulator 1; <I>SLC9A3R1</I>) together with sperm motility parameters and capacitation status to predict fertility/infertility in boars at the commercial level. Our data showed that among motility parameters and the capacitation status, hyperactivation (HYP) differed between high- and low-litter size boars. HYP showed a significant positive correlation (R = 0.468) with boar litter size. Simultaneously, the expression of <I>SLC9A3R1</I>, a gene important in sperm ion channel regulation, was significantly negatively correlated (R = −0.523) with boar litter size. Quality assessment revealed that both HYP and <I>SLC9A3R1</I> showed considerable sensitivity (71.43 <I>vs.</I> 100%), specificity (100 <I>vs.</I> 71.43%), and overall accuracy (90%) for predicting male fertility. Interestingly, the potential of <I>SLC9A3R1</I> expression to increase the average piglet number per breeding was higher (0.7 piglets) than that of HYP (0.5 piglets). Thus, measuring <I>SLC9A3R1</I> expression in spermatozoa may be a more accurate marker for evaluating male fertility/infertility than conventionally used motility parameters and capacitation status.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>SLC9A3R1</I> expression was negatively correlated with Yorkshire boar litter size. </LI> <LI> <I>SLC9A3R1</I> expression showed a remarkable increase of 1.5 (net increase in 0.7) piglets per insemination. </LI> <LI> <I>SLC9A3R1</I> expression in ejaculated spermatozoa may be a biomarker for predicting male fertility under field conditions. </LI> </UL> </P>

Optimized combination of multiple biomarkers to improve diagnostic accuracy in male fertility

Park, Yoo-Jin,Pang, Won-Ki,Ryu, Do-Yeal,Song, Won-Hee,Rahman, Md Saidur,Pang, Myung-Geol Elsevier 2019 Theriogenology Vol.139 No.-

<P><B>Abstract</B></P> <P>Artificial insemination is the general method of breeding for genetic improvement in offspring. However, almost half of the insemination cases fail to achieve full-term pregnancy, due to male infertility or subfertility. To maximize the success of insemination, accurate semen quality testing is required prior to insemination. Even though basic semen analyses have been used to provide preliminary information, it cannot fully identify the superior or inferior fertility bulls. Therefore, more powerful and easy to use methods for the prediction of male fertility are required, such as proteomic or microarray chips. During past decades, omics approaches have been developed and suggested the numerous fertility-related potential biomarkers. Our previous study identified the fertility related protein markers, enolase1 (ENO1), ATP synthase, H<SUP>+</SUP> transporting, mitochondrial F1 complex, beta subunit (ATP5B), voltage-dependent anion channel 2 (VDAC2), phospholipid hydroperoxide glutathione peroxide (GPx4), and ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2) in bovine spermatozoa. In the present study, we perform a marker combination assay using the western blot data of ENO1, ATP5B, VDAC2, GPx4, and UQCRC2 from 20 individual bull semen samples. And then, we identified the predictive ability of these markers for normal (non-return rate (NRR) ≥ 70%) and normal fertility (NRR<70%) in bulls. ENO1, a single protein marker, achieved an area under the curve (AUC) of 0.86 and 90% discriminatory power between normal and below-normal fertility bulls, with 90% sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Although no meaningful changes existed in overall accuracy (70–85%) to discriminate the normal and below-normal fertility between ENO1 single marker and combined marker panels, multiple marker combination methods using ENO1, VDAC2, GPx4, and UQCRC2 provided absolute sensitivity and NPV, with higher specificity (70%) and PPV (77%). ENO1 can be used as a fertility marker candidate, but there were limitations for providing absolute information about normal and below-normal fertility. Although the combined use of fertility-related markers cannot provide absolute accuracy, it can help in indicating below-normal fertility in bulls. These results may contribute to the maintenance cost in the animal industry, via selection of bulls with inferior fertility.</P> <P><B>Highlights</B></P> <P> <UL> <LI> ENO1 protein achieved an AUC of 0.86 and 90% discriminatory power between normal and below-normal fertility bulls. </LI> <LI> Multiple combination using ENO1, VDAC2, GPx4, and UQCRC2 provided absolute sensitivity and negative predictive value. </LI> <LI> Multiple marker combination may be used as a standard for artificial selection or improving average bull fertility. </LI> </UL> </P>

A proteomic study to predict litter size in boar spermatozoa

Sung-Jae Yoon,Myung-Geol Pang 한국발생생물학회 2014 한국발생생물학회 학술발표대회 Vol.2014 No.9