Background: Sperm quality and the number of sperm introduced into the uterus during artificial insemination (AI) are pivotal factors influencing pregnancy outcomes. However, there have been no reports on the relationship between sperm concentration at AI and sperm quality in Hanwoo cattle. In this study, we examined sperm quality and pregnancy rates after AI using sperm inseminated at different concentrations. Methods: We evaluated the motility, viability, and acrosomal membrane integrity of sperm at different concentrations (10, 15, 18, and 20 million sperm/straw) in 0.5-mL straws. Subsequently, we compared the pregnancy rates after AI with different sperm concentrations. Results: After freeze-thawing, sperm at the assessed concentrations showed similar viability and acrosomal membrane integrity. After AI, cattle in the 10 million group had significantly lower pregnancy rates compared to those in the 18 and 20 million groups. Conversely, there were no statistically significant variances observed between cattle in the 10 and 15 million groups. Conclusions: Sperm at concentrations of 10, 15, 18 and 20 million per straw exhibited comparable motility, viability, and acrosomal membrane integrity. However, a concentration of at least 18 million sperm per straw is required to achieve a consistent rate of pregnancy rate in Hanwoo cattle after AI.

본 연구는 동자개 정자의 동결보존을 위해 동결보존제의 최적 농도와 적정 희석액을 구명하여 정자를 최상의 상태로 보존하여 인공종자를 생산하는데 목적이 있다. 실험은 3종의 희석액(Ⅰ: 300 mM glycose, Ⅱ: Kurokura extender, Ⅲ: Li extender), 4종의 동결보존제(dimethyl sulfoxide, ethylene glycol, methanol and glycerol)와 4개의 동결보존제 농도(5, 10, 15, 20%)를 조합하여 대하여 조사하였다. 동결보존한 정자를 해동한 후 정자의 생존율과 정자활성지수로 동결보존 제의 효과를 평가하였다. 희석액 Ⅲ(Li extender)과 10과 15%의 methanol을 조합했을 때 동자 개 정자의 생존율과 정자활성지수는 각각 66.9 ± 8.7, 67.3 ± 13.1%과 2.6 ± 0.4, 2.6 ± 0.5로 다른 희석액과 동결보존제보다 높았다.

The striped fruit fly (SFF), Zeugodacus scutellata, is an agricultural pest species with a strong and rapid reproductive ability that can cause significant harm. To control the population of these kind of pests, the sterile insect technique (SIT) is being used as one of the effective methods. SIT involves the introduction of sexually transmitted factors that reduce the reproductive capacity of males. This study shows that knocking down the testis-specific serine/threonine protein kinase 1 (Zs-Tssk1) gene alters male fertility and male-initiated types of communication. Since Zs-Tssk1 influences the physiology of the testes, spermatogenesis is also affected, which in turn alters the lifespan of Zs-Tssk1 knock down group in comparison with the control. Based on these results, Zs-Tssk1 may be crucial in reproductive function, and its down-regulation may be helpful in controlling SFF through SIT.

In this study, an experiment was conducted in order to determine what cryopreservatives (CPVs) were more effective in supporting the motility and viability of sperm from experimental animals. The sperm of mice, rats, beagle dogs, and rabbits were frozen using different CPVs, including DMSO, TYB, and Sperm CryoProtec. The results from freezing the sperm of each laboratory animal in Sperm CryoProtec showed a high level of sperm motility and viability in sperm samples from mice, rats, and beagle dogs melted at the end of the first week. For rabbits, a high level of motility was observed in sperm thawed during the first week, whereas a high level of viability was observed in sperm thawed during the second week. The results of analysis of sperm motility and viability using different CPVs according to laboratory animals showed a significantly higher level of sperm motility (26.28%) and viability (36.20%) for mice in Sperm CryoProtec and the lowest levels of motility and viability were observed in DMSO (p < 0.05). Significantly higher levels of motility (27.94%) and viability (37.94%) were observed for rats in Sperm CryoProtec compared with TYB, which showed the lowest levels of motility and viability (p < 0.05). The study findings described above suggest that the selection of appropriate cryopreservatives is required for each experimental animal. This is because there are differences in the levels of sperm motility and viability of experimental animals depending on the CPVs that are typically used for freezing human sperm, including Sperm CryoProtec and TYB.

Endocrine-disrupting chemicals found in many commercial products may interfere with the normal functioning of the endocrine system and are unsafe because of their cumulative effect on the human body. However, little is known about the effects of combinations of endocrine-disrupting chemicals in humans. Methoxychlor and bisphenol A are toxic to male reproductive organs. Therefore, we studied the effects of methoxychlor and bisphenol A on male reproductive function. Male mice were divided into four treatment groups: control, 400 mg methoxychlor, 1 mg bisphenol A, and 400 mg methoxychlor + 1 mg bisphenol A/kg/day. Methoxychlor and bisphenol A were dissolved in sesame oil and acetone and administered orally for 4 weeks. After administration, the weight and histological changes in the testicles and epididymis, sperm count and health were observed biochemical tests and whole blood counts were performed. The results showed that the mice in the bisphenol A and methoxychlor + bisphenol A groups gained more weight than those in the control and methoxychlor group. The weights of the testes and epididymis were higher in the experimental groups than in the control. Sperm motility and progression were significantly reduced in the bisphenol A and methoxychlor + bisphenol A groups. Histological observation showed a reduced number of sperm, smaller seminiferous tubules, and destroyed lumen in the methoxychlor + bisphenol A group compared to the other groups. In conclusion, our study showed that methoxychlor and bisphenol A destroy male reproductive tissues and decrease sperm quality.

This study investigated the influence of sodium bicarbonate (NaHCO3) and progesterone on acrosome reaction and proportion of polyunsaturated fatty acid (PUFA) composition boar sperm. The sperm were diluted with semen extender and incubated with NaHCO3 and progesterone at 38℃, 5% CO2 for 6 h. Plasma membrane integrity and acrosome reaction were analyzed using SYBR14/propidium iodide (PI) and FITC-PNA/PI doubling staining method, and proportion of PUFA was analyzed using gas chromatography. In results, Plasma membrane integrity was significantly decreased in 50 mM NaHCO3 group and acrosome reaction was significantly increased by over the 100 mM NaHCO3 group compared to control group (p < 0.05). In addition, progesterone significantly increased decreased plasma membrane integrity at 100 mM progesterone and acrosome reaction at over the 5.0 µM progesterone (p < 0.05), but there was no difference among the 5.0 to 100 µM groups. PUFAs were significantly decreased in 100 mM NaHCO3 and 50 µM progesterone treatments compared to control group. In summary NaHCO3 and progesterone induce acrosome reaction and reduce PUFA composition in boar sperm, therefore, the results maybe help to understand basically knowledge for the acrosome reaction and PUFA composition in boar sperm.

Subgroups of sperm which share similar motility features documented in mammals indicate between-subject variations that might be related to fertilizing potential of the respective ejaculates. The objectives of this study were to define subpopulations of motile sperm in Gyr falcon semen using kinematic parameters driven by Computer Assisted Semen Analysis (CASA) and to investigate the subjectrelated variations in these subpopulations. A total of 24 fresh ejaculates from 6 falcons were used to assign each of the 20473 sperms into 3 subpopulations by a multivariate cluster analysis. The proportion of sperms in different sub-populations were compared among subjects by a generalized linear model and repeatability of sperm frequency in different subpopulations was investigated by corelation analysis. The resulting 3 categories of sperm indicated significant differences in all kinematic parameters (p < 0.05). Subpopulation 1 (15.91%) contained sperms with the highest velocity and progressiveness of movement trajectory while subpopulation 3 (6.4%) included the least progressively motile sperms. Proportion of rapid and medium progressive sperm were consistently higher in the ejaculate of three falcons compared to the two other birds which also had the highest proportion of slow non-progressive sperms (p < 0.05). Respective proportion of sperms in each subpopulations indicated significant repeatability over multiple measurements (p < 0.05). In conclusion, subpopulations of motile sperm in Gyr falcon can be identified using kinematic parameters generated by CASA. Individual differences in the proportion of these subpopulations might have potential application for identifying the males with higher fertilizing capacity.

Epididymal sperm cryopreservation provides a potential method for preserving genetic material from males of endangered species. This pilot study was conducted to develop a freezing method for tiger epididymal sperm. We evaluated post-thaw sperm condition using testes with intact epididymides obtained from a Siberian tiger (Panthera tigris altaica ) after castration. The epididymis was chopped in Tyrode's albumin-lactate-pyruvate 1x and incubated at 5% CO2, 95% air for 10 min. The Percoll separation density gradient method was used for selective recovery of motile spermatozoa after sperm collection using a cell strainer. The spermatozoa were diluted with modified Norwegian extender supplemented with 20 mM trehalose (extender 1) and subsequent extender 2 (extender 1 with 10% glycerol) and frozen using LN2 vapor. After thawing at 37℃ for 25 s, Isolate® solution was used for more effective recovery of live sperm. Sperm motility (computerized assisted sperm analysis, CASA), viability (SYBR-14 and Propidium Iodide) and acrosome integrity (Pisum sativum agglutinin with FITC) were evaluated. The motility of tiger epididymal spermatozoa was 40.1 ± 2.0%, and progressively motile sperm comprised 32.7 ± 2.3%. Viability was 56.3 ± 1.6% and acrosome integrity was 62.3 ± 4.4%. Cryopreservation of tiger epididymal sperm using a modified Norwegian extender and density gradient method could be effective to obtain functional spermatozoa for future assisted reproductive practices in endangered species.

The immunological sperm separation method is economical compared to the existing sorting method, and it is promising for the development of new technologies by reducing sperm damage. Wholemom (WM) is a sex-regulating protein that comprises on immunoglobulin G coupled with magnetic nanoparticles (MNPs) that responds to surface proteins derived from the Y chromosome in cattle. Y sperms are restricted in motility as the WM aggregates them, and the magnet could separate the non-aggregated cells. This study was conducted to investigate the effect of WM treatment on the characteristics of bull sperm. After treating sperm with WM and incubation for 6 h, the motility parameters including total motility, progressive motility, velocity average path, velocity straight line, amplitude of lateral head displacement, and linearity were significantly higher in the WM treatment group than in the control group (p < 0.05). Sperm viability and acrosome reaction rates were similar in both groups during each incubation period (p > 0.05). In conclusion, the immunological sperm sexing procedure using a monoclonal antibody conjugated with MNPs did not affect the characteristics of bull sperm. This study suggests that compared to other techniques, the immunological method for sperm sexing could classify sperm quickly and efficiently without the use of expensive equipment.

Sperm cryopreservation is an important method of assisted reproductive techniques and storing genetic resources. It plays a vital role in genetic improvement, livestock industrial preservation of endangered species, and clinical practice. Consequently, the cryopreservation technique is well organized through various studies, especially on Korean native cattle (Hanwoo). However, the cryopreservation technique of Korean native brindled cattle, which is one of the native cattle species in Korea, is not well organized. Therefore, it is necessary to develop a Supplementary Table technique for the cryopreservation of Korean native brindled cattle. For this purpose, it is important to first evaluate the quality of the currently produced cryopreserved sperm of Korean native brindled cattle. In this study, we randomly selected 72 individual Korean native brindled cattle semen samples collected from 8 different region research centers and used them to evaluate sperm functions. We focused on the quality evaluation of cryopreserved Korean native brindled cattle semen following the measurement of motion kinematics, capacitation status, intracellular ATP level, sperm motility, and cell viability. Then, the values of each of the eight groups were derived from various sperm parameters of nine individual samples, including sperm motility, kinematics, cellular motility, and intracellular ATP levels, which were used to compare and evaluate sperm function. Overall, differences in various sperm parameters were observed between most of the research centers. Particularly, the deviations of motility and motion kinematics were high according to the sample. Therefore, we suggest that it is necessary to develop a standard method for the cryopreservation of Korean native brindled cattle semen. We also suggest the need for sperm quality evaluation of the cryopreserved semen of Korean native brindled cattle before using artificial insemination to attain a high fertility rate.

북방전복 Haliotis discus hannai 웅성생식세포의 분화과정과 정자의 형태를 미세구조적으로 기 재하였다. 정자의 분화과정은 정원세포기, 정모세포기, 정세포기 및 정자기의 4단계로 구분하였 다. 정원세포기에서 정모세포기로의 분화과정은 형태학적 변화가 크지 않았다. 그러나 정자변 태과정 동안 염색질 응축, 핵의 형태 변화, 첨체와 중편 및 편모 형성 등의 급격한 형태학적 변 화를 나타냈다. 북방전복의 정자는 두부, 중편 및 미부로 구성되며, 두부의 길이는 약 5.3 μm로 전자밀도가 높은 핵과 총알형의 첨체로 이루어져 있었다. 중편은 기저체와 미토콘드리아로 구 성되어 있었으며, 기저체를 중심으로 5개의 미토콘드리아가 한 층으로 배열되어 있었다. 미부 의 횡단면은 "9+2"의 미세소관 구조를 보였다. 이러한 형태 및 구조적 특징은 북방전복의 정자 는 원시형(primitive type) 정자임을 보여주는 결과이다.

The aims of the present study were to confirm that regulation of the PA and environment via TGF-β regulation of sperm by Percoll-separated in porcine uterine epithelial cells. And, it was performed to identify the cytokines (TGF-β1, 2 and 3, TGF-β receptor1 and 2; interleukin, IL-6, IL-8) and PA-related genes (urokinase-PA, uPA; tissue- PA, tPA; PA inhibitor, PAI; uPA-receptor, uPAR) by spermatozoa. The experiment used porcine uterus epithelial cells (pUECs) and uterine tissue epithelial cells, Boar sperm were separated by discontinuous Percoll density gradient (45/90%), and tissues were co-incubated with spermatozoa, followed by real-time PCR. PA activity was measured of sperm by discontinuous Percoll density gradient (45/90%) for 24 hours. To measure viability and acrosome damage of sperm double stained propidium iodide (PI) and SYBR- 14 or FITC-PNA were used. In results, binding ratio of Percoll-separated sperm was found no differences, but sperms isolated from 90% Percoll layer reduced PA activity (p < 0.05). when co-cultured sperm selected Percoll in porcine uterus tissues epithelial cells, 90% layer sperm increased TGF-β R1, contrastively tPA and PAI-1 in comparison with control (p < 0.05). 45% sperm was decreased the expression of uPA (p < 0.05). TGF-β decreased PA activity in the supernatant collected from pUECs (p < 0.05). Especially, The group including uPA, PAI-1 were induce sperm intact, while it was reduced in sperm damage when compared to control (p < 0.05). Also, there was no significant difference group of tPA and tPA+I in the dead sperm and acrosome damage compared to control. The expression of tPA and PAI showed a common response. Percoll-separated spermatozoa in 90% layer reduced tPA and IL-related gene mRNA expression. Thus, Percoll-sparated sperm in 90% layer show that it can suppress inflammation through increased expression of TGF-β and downregulation of PA and IL in epithelial cells compared to 45% layer Percoll.

Amniotic membrane stem cells are considered as a good alternative to embryonic stem cells, but their use in clinical studies is still not common. Here, exosomes from canine amniotic membrane mesenchymal stem cells (cAmMSCexo) were used for dog sperm cryopreservation. Upon cryopreserved straws using cryoprotectant containing 0, 0.5, 1, or 2 μg/mL of cAmMSC-exo were thawed, motility and membrane integrity were analyzed. However, results showed no significant differences between the groups. We concluded that cAmMSC-exo with lower than 2 µg/mL have no effects on sperm cryopreservation, and further studies to get higher concentrations of cAmMSC-exo should be conducted for clinical application.

The effects of the number of frozen-thawed ram sperm per single and double intra-cervical artificial insemination (AI) on fertility in ewes were studied. A total of 89 non-pregnant ewes were synchronized for oestrus with two doses of 100 μg PGF2α (Cloprostenol) 9 days apart. The ewes were randomly assigned to one of four groups; D200 (n = 23; double AI with 200 × 106 sperm), S200 (n = 24; single AI with 200 × 106 sperm), D100 (n = 24; double AI with 100 × 106 sperm) and S100 (n = 18; single AI with 100 × 106 sperm). Ewes were inseminated within 12 to 18 h for single AI and, within 10 to 12 h and 16 to 18 h for double AI after the onset of oestrus. The onset of oestrus ranged from 28 to 76 h (54.33 ± 1.28 h). The high percentage (29.2%) of ewes showed oestrus between 51 to 60 h. The non-return rates were highest in group D200 (56.5%) and differed significantly (p < 0.05) from group S100 (11.1%). No ewes were pregnant in group S100, and the pregnancy rates among the remaining groups did not differ. The mean gestation period was 152.8 ± 0.5 days and no difference was observed among the groups. The lambing and multiple birth rates were 100% in group D200. The single and twin lambing was highest in group D100 (33.3%) and group D200 (83.3%), respectively. Only one triplet lambing and the highest lambing size (2.2 ± 0.2) was recorded in group D200. In conclusion, double AI with 200 × 106 sperm showed comparatively most practical for achieving high pregnancy rates and lambing performances in Bangladeshi ewes under field conditions.

We observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. According to the results from each freezing extender, the sperm membrane integrity (HOST: Hypoosmotic Swelling Test) analysis in TCGGD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Glycerol 3%, Dimethylsulpoxide 3.5 M) is 59.8 ± 0.7, TCGSD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Sucrose 0.1 M, Dimethylsulpoxide 3.5 M) is 59.3 ± 0.5 were significantly higher (p < 0.05) among the experimental groups. And MMPs analysis result, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of active MMP-2 was the highest in sperms frozen in TCGSD and TCGD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Dimethylsulpoxide 3.5 M), Meanwhile, sperms from the TCGGD and TCGED (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Ethylene glycol 3%, Dimethylsulpoxide 3.5 M) group showed lower level of active MMP-2 expression. Together, these results indicate that adding glycerol or sucrose to the sperm freezing buffer would not only suppress MMPs expression but also minimize DNA fragmentation, providing a mean to improve the success rate in the in vitro manipulation of rabbit sperms. Therefore, these results suggest that TCGGD or TCGSD extender method for freezing-thawing of rabbit sperm increased the viability after thawing.

This study was conducted to analyze the specific genes associated with sex-determination in Korean native cow. The highly organized spermatogenesis requires accurate spatial and temporal regulation of gene expression, which is governed by transcriptional, post-transcriptional, and epigenetic processes. Recently, farmers have been interested in determining the sexual identity of the calves in their farm. We analyzed the sperm of Korean native and Holstein cows, which were supplied from Hanwoo Improvement Center. We evaluated sperm motility and expression of sperm-specific genes after treating semen with both male- and female reagents. Sperm motility in Korean native cows decreased by approximately 10% in the first 30 minutes after treatment with sex-determination reagent. However, sperm motility of Holstein cows decreased to 60-70% after 15 minutes and to 20-30% after 30 minutes. We selected six specific genes expressing in the spermatozoa to analysis the gene expression level. The Real-time PCR results suggest that the selected genes (Gimap4, Tmeff1, Rac2, Abi2, Rac1, and Clu) were highly expressed in the group treated with the male reagent compared to the group treated the female reagent and to the untreated-group (control). In the present study, we suggest that the selected genes play a pivotal role in sex-determination.

The purpose of this study was to evaluate the effect of addition of ethylene glycol, glycerol and sucrose to TCG (Tris, Citric Acid, Glucose, Egg Yolk) and DMSO Frozen. The extender containing Egg yolk concentration (10%, 20%) affects viability and acrosome morphology of rabbit sperm. Sperm viability was then assessed for the freezing extenders TCGD (Tris + Citricacid + Glucose + DMSO), TCGED (Tris + Citricacid + Glucose + Egg yolk + DMSO), TCGGD (Tris + Citricacid + Glucose + Glycerol + DMSO) and TCGSD Tris + Citricacid + Glucose + Sucrose + DMSO) during thawing at 38oC. for 20 seconds, respectively. TCG + 10% egg yolk (viability: 77.0 ± 0.8, NAI: 73.3 ± 0.9) was significantly (sperm viability and normal acrosome interaction (NAI)) higher than TCG + 20% egg yolk (70.7 ± 1.1, 70.0 ± 0.9) in the sperm normalcy analysis according to the yolk concentration. TCGGD (53.4 ± 0.1, 62.3 ± 0.4), TCGSD (61.3 ± 0.0, 67.1 ± 0.1) sperm viability and normal acrosome interaction (NAI) in frozen spermatozoa are TCGD (46.4 ± 2.8 and 56.3 ± 1. 4) and TCGED (23.0 ± 1.1 and 54.6 ± 1.4) extenders was thawed at 38oC for 20 seconds. According to the results from each frozen bulking agent, sperm membrane integrity by hypotonic swelling test (HOST) analysis in TCGGD (59.8 ± 0.7), TCGSD (59.3 ± 0.5) was significantly high compared to other experimental groups (p < 0.05). In conclusion, these results suggested that TCGGD and TCGSD extenders enhance survivability of rabbit sperm after frozen-thawing.

This study was conducted to find out the effect that κ-Carrageenan has on the properties of dog sperm when it was added to the cryoprotectant. Extender basically was contained 1.21 g Trizma base, 0.67 g citric acid, 0.4 g glucose, 0.03 g penicillin G, 0.05 g streptomycin sulfate. Extender1 was added with 0.1%, 0.2%, 0.3%, and 0.5% carrageenan, while extender2 was supplemented with glycerol. After freezing-thawing, the motility, viability, acrosome integrity, apoptosis, and ROS (reactive oxygen specifications) of sperm were measured to analyze the effects of the supplementation of carrageenan. Total Motile (TM), Rapid Progressive Motile (RPM), Medium Progressive Motile (MPM), and Immotile were measured through the CASA system after thawing in 37 degree water. Extender with 0.2% κ-carrageenan (64.26 ± 0.49) was significantly higher than control (40.24 ± 8.27) (p < 0.05). RPMs of extender with 0.1%, 0.2% κ-carrageenan (57.64 ± 6.34, 56.47 ± 1.35) were significantly higher than the other groups (p < 0.05). Acrosome integrity was measured by dyeing to PSA-FITC with an epifluorescence microscope. Normal acrosome ratio of extender with 0.5% κ-carrageenan (61 ± 8.03) was higher than the other groups (p < 0.05). Apoptosis was measured with a FACSCalibur flow cytometer using FITC (FITC Annexin V Apoptosis Detection Kit). Treated groups of κ-carrageenan of 0.1% (0.81 ± 0.05), 0.2% (0.85 ± 0.05) were significantly higer (p < 0.05) than control. Modified SYBR/PI staining was used for determination of viability and DCF staining was used for evaluation of ROS. Viability and ROS were not significantly different from other groups. In conclusion, adding a certain concentration of carrageenan to the extender of cryopreservation, carrageenan contributes to the improvement of the sperm motility, acrosome integrity and prevention of apoptosis.