• Animal Bioscience
• /
• 제36권12호
• /
• pp.1796-1805
• /
• 2023

Objective: This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers. Methods: The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) (79.04%; n = 4), and low fertility (LF) (65.84%; n = 4). mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate. Results: Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality. Conclusion: HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.

• 한국가축번식학회지
• /
• 제26권2호
• /
• pp.119-124
• /
• 2002

본 연구는 돼지에서 동결정액을 이용한 번식능력을 개선하기 위한 동결정액 포장재료의 효과를 구명하기 위하여 실시하였다. 본 시험에는 축산기술연구소 종축개량부 (충남, 성환)의 인공수정센터에서 사육중인 종모돈이 사용되었다. 기존의 돼지 동결정액 포장방법인 maxi-straw 동결정액 포장방법과 5$m\ell$빈 cryogenic-vial 및 aluminum-pack 포장방법을 비교한 결과 cryogenic-vial로 포장하여 액체질소 상단 15cm에서 동결한 후 52$^{\circ}C$ water bath에서 190초 융해한 방법이 기존의 maxi-straw 방법과 비슷한 결과를 나타내었다. Cyogenic-vial 포장방법의 동결-응해 방법을 설정하기 위하여 융해시간을 달리하여 시험한 결과 액체질소 상단 15cm에서 동결하고 52$^{\circ}C$에서 190초 간 융해하였을 때 정자운동성이 120초 및 150초 응해시 보다 우수하였다 (P<0.05). 그러나 정상첨체비율은 응해시간 간에 차이가 없었다. 52$^{\circ}C$에서 45초간 응해 한 maxi-straw 포장방법과 52$^{\circ}C$에서 190초 융해한 cryogenic-vial 포장방법간에 정액성상을 비교한 결과 총정자운동성과 정자의 빠르기는 maxi-straw가 우수하였다 (P<0.05). 그러나 직진성과 정상첨체비율은 두 포장방법간에 차이가 없었다. 동결정액 포장방법별 인공수정시 번식성적은 maxi-straw 동결정액이 cryogenic-vial 동결정 액보다 수태율, 분만율, 그리고 산자수가 높았으나 통계적 유의차는 인정되지 않았다. 이상의 결과를 종합해 보면 cryogenic-vial 포장 방법의 동결 및 음해방법을 좀 더 연구개발하면 기존의 maxi-straw포장방법을 대체하여 실용화 할 수 있을 것으로 사료된다.

• 한국산학기술학회논문지
• /
• 제21권3호
• /
• pp.199-205
• /
• 2020

정액의 동결보존은 인공수정을 통한 동물 번식에 유용한 것으로 알려져 있지만 동결-융해된 돼지 정액의 사용은 저온손상 때문에 제한된다. 최근에는 이를 보완하기 위해 다양한 첨가제 연구가 진행 되고 있다. 그 중 항산화제는 정자의 동결 융해과정에서 정자 세포막의 지질과산화를 억제시켜 정자의 생존성과 운동성을 개선시킨 다고 알려져 있다. 본 연구의 목적은 동결보존액에 대한 MitoTEMPO(미토콘드리아 표적 항산화제) 첨가가 돼지 동결-융해 정자의 운동학적 특성에 미치는 영향을 평가하는 것이다. 성숙한 Duroc종 수퇘지로부터 정액샘플을 채취하였으며, 다양한 농도의 MitoTEMPO (0, 0.5, 5, 50 및 500 μM)를 lactose-egg yolk 동결보존액에 첨가하여 정액을 동결하였다. 동결-융해 후 정자의 운동학적 특성들은 정자자동분석기 (CASA; computer-assisted sperm analysis)를 이용하여 분석하였다. 그 결과, 동결용 보존액에 5 및 50 μM (50.46±2.71%, 46.96±2.66%) MitoTEMPO 첨가 시 500 μM 처리구(35.40±2.95%)에 비해 유의적으로 높은 정자 운동성을 나타냈다(P<0.05). 그렇지만, 운동성을 제외한 다른 운동학적 특성에서는 유의적인 차이를 보이지 않았다. 결론적으로 동결용 보존액에 대한 MitoTEMPO 첨가는 동결-융해 돼지 정자의 운동성에 긍정적인 영향을 미치는 것으로 사료된다.

• 한국가축번식학회지
• /
• 제26권3호
• /
• pp.229-234
• /
• 2002

본 연구는 불임견의 번식장애를 해결할 목적으로 개 정소상체로부터 채취한 정액을 tris-buffer로 원심분리하여 정장을 제거한 RSP-T 희석 정자의 일반성상과 동결방법 및 glycerol 농도가 동결융해 후 생존성에 미치는 영향과 난자와 정소상체 정자를 매정시켰을 때 정자침입율을 조사하기 위하여 수행하였다. 1. WES, RSP-S 및 RSP-T 희석 정소상체 정액의 정자수는 각각 4.25 $\pm$ 0.25(X$10^{6}$ Cells/$m\ell$), 3.85 $\pm$ 0.20(x$10^{6}$cells/$m\ell$), 4.05 $\pm$ 0.28(X$10^{6}$cells/$m\ell$)이었고, 활력은 50.55 $\pm$ 2.75%, 67.25 $\pm$2.55%, 78.75$\pm$3.55%이었고, 기형정자율은 49.45$\pm$2.25%, 37.75 $\pm$ 2.10%, 24.25 $\pm$ 1.55%이었다. 2. RSP-S와 RSP-T 희석 정소상체 정액을 완만 동결을 했을 때 생존율은 각각 35.00 $\pm$2.35%, 45.50 $\pm$2.15%와 초급속동결시의 생존율은 16.50 $\pm$ 3.55%, 22.55 $\pm$ 3.95%이었다. RSP-T 희석 정소상체 정액을 동결시 내동제에 glycerol 농도를 2.0~8.0% 첨가하여 동결했을 때 생존율은 각각 9.25 $\pm$ 1.55%~17.50$\pm$2.50%로서 신선정액의 25.50% 4.50%~34.00$\pm$5.15%에 비해 낮은 생존율을 나타냈다. 3. 동결 융해한 정소상체 희석정자의 평균 수정 능 획득 율과 첨체반응 및 생존정자수는 각각 13.00 $\pm$ 2.35%, 3.55 $\pm$ 0.85%, 15.50 $\pm$ 1.90% 였고, 신선 정소상체 정액의 45.25$\pm$5.75%, 7.06 $\pm$0.25%, 49.20$\pm$6.80%이었다. 신선 및 동결 수정 능 획득 정자를 난자와 매정시켰을 때 난자내 정자의 침입율은 각각 39.25 $\pm$4.72 %와 34.24$\pm$3.93%였고, 난자당 정자의 침입 수는 각각 1.30$\pm$0.33개, 1.10$\pm$0.50개이었다.

• 한국동물생명공학회지
• /
• 제35권4호
• /
• pp.307-314
• /
• 2020

In the present study, we examined the effect of straw size on spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. Hanwoo semen was collected from three bulls and diluted with an animal protein-free extender, divided into two groups, namely, 10 million spermatozoa in 0.25 mL and 20 million spermatozoa in 0.5 mL straw, and cryopreserved. In Experiment 1, the motility and motility parameters of the frozen-thawed spermatozoa were evaluated. After freezing-thawing, the spermatozoa motility parameters fast progressive, straight line velocity, and average path velocity were compared between the 0.25 mL straw and 0.5 mL straw groups. They were 35.2 ± 1.0 and 32.3 ± 0.7%, 34.6 ± 0.7 and 31.8 ± 0.5 μm/s, 51.4 ± 1.3 and 47.1 ± 1.1 μm/s, 0.25 mL straw and 0.5 mL straw groups, respectively. In Experiment 2, the viability, acrosome membrane integrity, and mitochondrial membrane potential of the frozen-thawed spermatozoa were assessed. After freezing-thawing, the percentages of spermatozoa with live, intact acrosomes and high mitochondrial membrane potential were compared between the in 0.25 mL straw and 0.5 mL straw groups. They were 48.0 ± 2.6% and 35.6 ± 2.8% between the 0.25 mL straw and 0.5 mL straw groups. In Experiment 3, the plasma membrane integrity of frozen-thawed spermatozoa was compared. After freezing-thawing, the plasma membrane integrity was higher for the in 0.25 mL straw group than the 0.5 mL straw group. They were 62.0 ± 2.2 and 54.1 ± 1.3% between the 0.25 mL straw and 0.5 mL straw groups. In conclusion, our results suggest that freezing semen in 0.25 mL straw improves the relative motility, viability, and acrosomal, mitochondrial membrane potential, and plasma membrane integrity of Hanwoo bull spermatozoa.

• Reproductive and Developmental Biology
• /
• 제35권1호
• /
• pp.61-65
• /
• 2011

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on motile sperm recovery rate and motion kinematics. Frozen semen samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk-Tris-glycerol extender) were thawed in $37^{\circ}C$ water bath for 1 min. After thawing, the mixed semen samples were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities ($300{\times}g$ and $700{\times}g$) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Motile sperm recovery rate and motion kinematics were evaluated by computer assisted sperm analyzer using Makler counting chamber. Sperm motility (%) and motile sperm recovery rate showed similar pattern in all treatment groups. However, sperm motility (%) and motile sperm recovery rate were highest at $700{\times}g$ for 30 min through a discontionous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll. There were no significant differences in motion kinematics after various Percoll washings. These results suggest that force of centrifugation, centrifugation time, and Percoll volume significantly affect motile sperm recovery rate.

• Asian-Australasian Journal of Animal Sciences
• /
• 제23권10호
• /
• pp.1276-1281
• /
• 2010

The present study was conducted to investigate the effects of butylated hydroxytoluene (BHT) supplementation on diluted, cooled and frozen-thawed ram spermatozoa. After primary evaluation of collected ejaculates, only semen samples with motility of more than 70% and sperm concentration higher than $3{\times}10^3$ sperm/ml were used for cryopreservation. The selected semen samples were then pooled and diluted 1:4 with Tris Citrate Fructose Yolk (TCFY) extender supplemented with different concentrations of BHT (0.5, 10, 2.0 and 3.0 mM). As the control, semen was diluted and frozen in the diluent without BHT. Motility, progressive motility, viability, membranes and acrosome integrity were evaluated after dilution (part 1), cooling (part 2) and freezing and thawing (part 3). The results of the first part of the experiment showed that there were no significant difference between treatments in the motility, progressive motility, viability, membranes and acrosome integrity of spermatozoa, but the results with 2.0 mM BHT were slightly better than obtained with other levels of BHT and control extender. Significantly better results (p<0.05) were observed in the second part of the experiment for cooled spermatozoa characteristics, when extender was supplemented with 2.0 and 3.0 mM BHT. Furthermore, the results obtained in the third part of the experiment indicated that, after freezing and thawing, all evaluated semen characteristics were improved significantly (p<0.05) by increasing BHT levels, with the best results obtained for extender containing 2 mM BHT. Comparison of these results with those of control diluent, the effects of supplementation were significantly (p<0.01) better. However, the higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of spermatozoa compared to extender containing 2.0 mM BHT. In conclusion, the results obtained in this study showed that the semen quality of rams was improved when BHT was added to extender used before the freezing process.

• 한국수정란이식학회지
• /
• 제28권3호
• /
• pp.297-302
• /
• 2013

Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of genetically elite populations. Castration, catastrophic injury, sudden death or any other event that makes semen collection or mating impossible may prematurely terminate a stallion reproduction. Stallion epididymal spermatozoa vary widely in the loss of progressive motility, acrosomal integrity, and viability during freezing and thawing. The objective of this work was to investigate the effect of (1) freezing package types on cryopreservation efficiency, (2) thawing temperatures (37, 56 or $70^{\circ}C$) on Computer Assisted Sperm Analysis (CASA) parameters and (3) post-thawing incubation time (0, 1, 2 or 4h) on castrated stallion epididymis. Post-thawed sperm motility ranged between 59.69% and 64.28% ($56^{\circ}C$ and $37^{\circ}C$) in various thawing temperatures. When stallion epididymis sperm was frozen, straw was better than freezing tube on VCL (Velocity of Curvilinear Line) and VAP (Velocity of Average Path) parameter. Higher percentage of motility was observed at $37^{\circ}C$ thawing temperature even though no significant difference was observed among various temperatures. The motility, VCL, ALH (Amplitude of Lateral Head displacement), VAP, BCF (Beat-Cross Frequency) and STR (Straightness index) parameter of post-thawed sperm were significantly decreased by increasing the incubation time for all thawing temperatures. The present study showed that type of freezing package (Straw vs. Freezing tube) was not significantly different on cryopreservation efficiency. Furthermore, stallion epididymal spermatozoa frozen-thawed at $37^{\circ}C$ for 1 min resulted the highest proportion of motility and velocity movement. In addition, motility and viability of frozen-thawed stallion epididymal spermatozoa were also decreased by incubation.

• 한국동물생명공학회지
• /
• 제35권2호
• /
• pp.183-189
• /
• 2020

The pregnancy rate in indigenous ewes inseminated with frozen-thawed Suffolk semen following natural and synchronized estrus was determined. The serum Progesterone and Estrogen concentration and vaginal electrical resistance (VER) of ewes at the time of Artificial Insemination (AI) were observed as successful pregnancy determinants. 21 healthy ewes were selected for this experiment during January-April, 2017. 10 ewes were inseminated in natural estrus. Whereas, 11 ewes were inseminated after estrus synchronization using intravaginal sponges containing 60 mg medroxyprogesterone acetate. Trans-cervical Al (TCAI) was performed in all ewes within 12-16 hours of observed heat. Prostaglandin E₁ analogue impregnated vaginal sponge was used for cervical relaxation 6-8 hours before insemination. Pregnancy was diagnosed through trans-abdominal ultrasonography after 40 days of AI. The pregnancy rate of ewes in synchronized estrus was higher (54.5%) than in natural estrus (30%). Higher serum Progesterone level (0.90 ± 0.02 ng/mL) and significantly (p < 0.001) lower VER (257.78 ± 10.11 ohm) were observed at the time of AI in ewes becoming pregnant. Results suggest that higher Progesterone concentration and lower VER could be considered as pregnancy indicators. Oestrous synchronization could be implemented to increase the pregnancy rate in ewes.

• 한국임상수의학회지
• /
• 제19권1호
• /
• pp.73-79
• /
• 2002

To investigate the usability of frozen canine embryos for embryo transfer in the dog, 19 donors, 3 recipients, and 6 male dogs were used for the experiment. Natural mating or artificial insemination was performed for breeding the bitches in natural estrus. Vaginal smear test along with progesterone titre test were performed to detect the appropriate mating time and the bitches were bred twice during 3-6days following LH surge. Embryo collection was done on 8, 9-11, 12-13 days after the second mating to collect morula and blastocyst. Embryos were frozen using a programmable freezer and preseued in LNE tank. Embryos were thawed in 37$^{\circ}C$ water for 15 seconds and transferred into each uterine horn within 30 minutes. Embryos were collected from 13 bitches of 19 donors(68.4%) and the collected embryos were from between 9 and 13 days after 2nd mating. Embryos were produced both by natural mating(60.0%, 9115) and AI with frozen semen(100.0%, 4/4). Embryos were collected from the donors weighed between 2.5 and 30 kg and their age was from 1.5 to 3 years. 52 embryos were collected from 13 donors and the mean number of embryos was four. The stage of embryos was from 2-cell to gastrula and morulae were colledted mostly from 10 to 11 days after 2nd mating. Embryos were collected evenly from each uterine horn and the rate of embryo collection for the number of corpus luteum was 83.9%. Embryos were transferred to 3 recipients(morula 8, blastocyst 1, gastrula 8), however, no offspring was produced.