• Journal of Embryo Transfer
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• v.28 no.3
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• pp.193-198
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• 2013

This study was designed to evaluate the effect of L-cysteine on sperm characteristics and oocyte cleavage in vitro in Korean native cattle. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk and/or 0, 5, 10 and 20 mM L-cysteine before cryopreservation. The viability in frozen-thawed sperm were estimated by SYBR14/PI double stain, acrosome damage with FITC-PNA, mitochondria intact with Rhodamin123 and hydrogen peroxide($H_2O_2$) level with carboxy-DCFDA by flow-cytometry. The developmental capacity was also assessed with cleavage rates in oocytes fertilized in vitro by frozen-thawed sperm. In results, the sperm viability was significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). In addition, acrosome damage was significantly decreased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). The mitochondria intact was also significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). On the other hand, the cleavage rates were significantly increased in 0 mM, 5 mM and 10 mM groups than 20 mM concentration of L-cysteine (p<0.05). The oocyte degeneration of oocytes were significantly decreased in 0 mM, 5 mM and 10 mM groups than in 20 mM L-cysteine group (P<0.05). However, there are no significantly differences among the L-cysteine treatment groups. We suggest that concentration of 10 mM L-cysteine have beneficial impact for sperm cryopreserved in Korean native cattle. This result also could be recommended for artificial insemination program if supported by an improvement in the fertility results and required further study.

• Journal of Embryo Transfer
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• v.16 no.1
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• pp.35-40
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• 2001

Artificial insemination (AI) with frozen or cooled semen is widely used in commercial fields of cattle and pig. Little is known about characteristics of canine sperm after freezing or cooling. For both practical and commercial goal, the canine semen treated with cooling and freezing should be carried out to exam the fundamentals, including sperm motility, survivability and fertilizing capacity. The aim of this study, thus, was to identify the effects of extended exposure to 4$0^{\circ}C$ on canine semen by motility, survivability, acrosomal changes following different duration. Fifteen ejaculates collected by digital manipulation twice per week from 3 dogs (Shih-Tzu) were divided to 16 aliquots after adding Tris-egg yolk (TE) buffer formulated by our laboratory, and cooled from 37 to 4$^{\circ}C$, by ramp rate of 0.6$^{\circ}C$/min. Each sample was evaluated by their motility, survivability and the acrosomal status at 0h (control), 2h, 12h and 1 d~10 d, respectively. The motility of spermatozoa was graded to 6 levels using the modified method of Seager. The survivability of sperm was assessed using an epifluorescence microscope after Fert/Light (Mole-cular Probes Inc.) staining. To estimate the proportion of the spermatozoa of intact acrosome, 200 spermatozoa were assessed in randomly selected fields, using epifluorescence microscope after FITC/PSA (Sigma) staining. At 2 h after cooling, the motility of most spermatozoa were assessed to be grade 0 and 1. At 12 h, high number of sperm were in grade 0 to 1, however, it was significantly (P<0.05) lower than that of 2 h. From 1 d to 4 d, ~50% of sperm was assessed to grade 0 to 1. On day 7, a little sperm were in grade 0 to 1. No sperm showed motility on day 10. Sperm motility was rapidly reduced by the percent of 10% of grade 0 to 1. From 2 h to 6 h, the number of live sperm was 90% and the sperm chilled for 10 days lived>50%. Acrosomal intact of spermatozoa exposed to 4$^{\circ}C$ for 2 h was 51%, supposed the sperm of control was 100%. Our results suggest that 1) this is easy to transfer and preservation for short periods 2) AI can be used by semen chilled for 6-Day.

• Korean Journal of Animal Reproduction
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• v.23 no.3
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• pp.191-203
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• 1999

The objectives of this study were to investigate 1) the effects of Selenium(Se), Vitamin E (Vit. E) or recombinant Bovine Somatotropin(rBST) administration on fresh and frozen/thawed semen characteristics and 2) the effect of taurine on frozen/thawed semen characteristics in Hanwoo sires Hanwoo sires were randomly assigned to five groups (1. control, 2. rBST, 0.09mg/kg body weight (BW), 3. Vito E 1,500IU/kg BW, 4. Se 0.l mg/kg BW, 5. Vit. E 1,500IU plus Se 0.1 mg/kg BW). The administration of Se, Vit. E and rBST for each experimental group were given 6 times at 15 days interval by intramuscular injection. The administration of Se, Vit. E or rBST in Hanwoo sires didn't affect semen volume and pH values, but sperm viability was significantly increased comparing to the control group. Also, frozen/thawed semen analysis showed that the sperm viability increased, but any other effects were not found in total sperm :lumber, motility and abnormality among treatments. The addition of taurine in semen freezing extender had a beneficial effects on frozen/thawecl semen characteristics in all groups. The administrations of rBST, Vit. E and Se did not affect the sperm capacitation and acrosome reaction, either the ratio of F pattern(uncapacitated and acrosome intact sperm) or AR pattern(capacitated and acrosome-reacted sperm), but the ratio of B patten(capacitated and acrosome intact sperm) of treatment groups was significantly higher than that of control group, These results indicated that the viability, motility and quality of semen in Hanwoo sires were slightly increased by the injection of rBST, Vit. E and Se, and the addition of taurine in semen freezing extender were also increased the semen characteristics after thawing.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.307-314
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• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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• pp.351-358
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• 2007

Carbohydrate-protein interactions are known to be important in gamete interactions. Several evidence indicated that a fucose-containing sulfated polysaccharide fucoidan was potential inhibitor of fertilization in vitro and thus fucose seemed to be part of the recognition signal of gamete interaction in mammals. In recent investigation we found that ${\alpha}$-L-fucosidase activity was present in boar spermatozoa and it was related to sperm binding to and penetration into zona pellucida (ZP) in vitro. The objective of this study was to determine the effects of monosaccharide L-fucose and polysaccharide fucoidan on sperm ${\alpha}$-L-fucosidase activity and relation to sperm-oocyte interaction in pig. Results indicated that the activity of sperm ${\alpha}$-L-fucosidase was largely inhibited (62%) when sperm suspension was treated with monosaccharide L-fucose. It also significantly inhibited the number of sperm binding to ZP (32%) and penetration into zona-intact oocytes (72%), but did not inhibit penetration into zona-free oocytes when fertilization medium contained L-fucose. The chlorotetracycline (CTC) assessment showed that L-fucose did not affect induction of sperm capacitation and acrosome reaction. In contrast, the activity of sperm ${\alpha}$-L-fucosidase was not inhibited when sperm suspension was treated with polysaccharide fucoidan but sperm-ZP binding was greatly inhibited (85%) and completely blocked sperm penetration into zona-intact or zona-free oocytes. The CTC assessment showed that fucoidan increased the F pattern and decreased the AR pattern sperm. These results suggested that the different inhibitory mechanisms were present between monosaccharide L-fucose and polysaccharide fucoidan on sperm-oocyte interaction, the inhibition effect of ${\alpha}$-L-fucose on sperm binding and penetrating into ZP caused sperm ${\alpha}$-L-fucosidase inhibited by ${\alpha}$-L-fucose.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.297-306
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• 2020

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.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.435-439
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• 2011

Sildenafil citrate (SIL) a phosphodiesterase 5 inhibitor (PDE5I) has been used for long time as a first line oral drug for erectile dysfunction. Though it has beneficial effects on erectile organ it also has some adverse effects in other cells and/or tissues related to reproductive system when exposed to longer duration. The objective of the present study is to evaluate the long term effect of SIL on sperm parameters in Wistar albino rat. The animals are divided into two groups, for group I - rats were treated with saline (vehicle alone) and group - II oral administration of 5 mg/kg b.w. of SIL was administrated orally once in a day for 120 days. At the end of the trial period animals were sacrificed and epididymal sperm were subjected to various analysis. Results showed significant reduction in sperm count, motility, viability and morphologically intact sperm in long term PDE5I exposed animals when compared to control. Acrosomal status and fertility test also showed significant reduction in long term PDE5I exposed animals. The present study clearly indicated that long term SIL has shown to induce alteration in sperm quality and quantity, leading to decline in fertility rate. Indicate that SIL impinge on spermatogenesis as well as epididymal function. Understanding the molecular down-stream events involved in long-term exposure to PDE5 inhibitor can be valuable to supervise on related infertility issues and to suggest corrective measures.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.237-245
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• 2010

Only an optimum number of viable spermatozoa in a frozen-thawed insemination dose can ensure conception at artificial insemination (AI). We report here the percentages of normal, abnormal and viable spermatozoa present in the frozen-thawed semen of 20 Black Bengal bucks used for commercial AI. Bucks in this experiment were of 19.3~46.1 months old and 25~42 kg body weight. Four semen straws (0.25 ml) from each buck were collected for evaluation of their kinetic parameters. Scrotal circumference was measured by using a scrotal tape, sperm motility was estimated on eye estimation and sperm concentration was determined by using a haemocytometer. Sperm morphology was studied in paraformaldehyde fixed spermatozoa under differential interference contrast (DIC) microscope. To determine the proportion of live (plasma membrane intact) spermatozoa, semen was stained with SYBR-14 and propidium iodide and examined under fluorescent microscope. Scrotal circumference, post-thaw sperm motility, sperm concentration per insemination dose and proportion of normal spermatozoa were $21.5{\pm}0.7\;cm$, $43.5 {\pm}5.4%$, $83.5{\pm}6.7$ million and $88.3{\pm}4.1%$, respectively. The percentages of spermatozoa with head shape and acrosome abnormalities were lower ($2.7{\pm}1.1$ and $1.4{\pm}1.3$, respectively), whereas higher percentages of abnormalities ($7.0{\pm}1.8$) were observed in mid piece and tail portion. The proportion of live spermatozoa was $28.5{\pm}5.4$. It is concluded that although a good number of morphologically normal spermatozoa are present in the insemination dose, the proportion of live spermatozoa is low, which warrants further improvements of buck semen freezing procedures to ensure good quality at AI.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.45-50
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• 2012

The objective of this study is to estimate the effect of adding TES to LEY and FGE freezing extender for the sperm viability, acrosomal morphology and DNA fragmentation from miniature pig sperm, we evaluated sperm characteristics in TFGE, TLE and LEY with various thawing condition ($37^{\circ}C$ for 20 sec, 45 sec and $75^{\circ}C$ for 5 sec, respectively), and in different concentration of glycerol at 1%, 1.5%, 3%. The sperm viability and normal acrosome intact(NAI) in TFGE (Viability : $60.3{\pm}2.4$, NAI : $58.6{\pm}2.2%$), TLE ($61.3{\pm}2.4$, $62.2{\pm}2.2%$) extender significantly(p<0.05) increased than that in LEY ($50.2{\pm}2.4$, $54.5{\pm}2.2%$) extender thawed at $75^{\circ}C$ for 5 sec. According to the results from glycerol concentration, the viability and NAI of miniature pig sperm in 1.5% glycerol TLE ($66.1{\pm}3.2$, $66.2{\pm}1.0%$) was highest among the experimental groups. In accordance with this, DNA fragmentation rates was the lowest in TLE ($43.3{\pm}0.5%$) while that in LEY ($63.5{\pm}2.3%$) is the highest. Therefore, these results suggest that TLE extender method for freezing- thawing of miniature pig sperm increased the viability after thawing.

• Animal Bioscience
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• v.34 no.5
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• pp.844-854
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• 2021

Objective: The potential of extra virgin olive oil (EVOO), betaine (BET), and ginger (GIN), as natural antioxidants, in reducing negative effects of heat stress on physiological responses, antioxidant capacity, semen quality and fertility of bucks under heat stress were investigated. Methods: Forty adult Animal Production Research Institute line rabbit bucks were distributed randomly into four experimental treatments of ten rabbits each. The first treatment was fed the commercial pellet diet (CPD) without supplementation and served as a control. The other three treatments were fed CPD supplemented with EVOO (300 mg), BET (1,000 mg), and GIN (200 mg) per kg diet for 3 consecutive months during the summer season. Results: Supplementation of EVOO, BET, or GIN improved (p<0.05) the sexual desire, progressive motility, vitality, intact acrosome and membrane integrity, sperm cell concentration, sperm outputs and fertility. Seminal plasma total proteins, globulin, total antioxidant capacity, glutathione and glutathione S-transferase, and initial fructose increased (p<0.05), while total lipids, aspartate and alanine aminotransferases and malondialdehyde decreased (p<0.05) compared with the control. In comparing the natural antioxidants treatments, GIN evoked the largest improvement. Conclusion: The inclusion of GIN (200 mg/kg diet) appeared to improve the sexual desire, semen quality and oxidative stress of bucks. This may be a beneficial supplement for the management of rabbit bucks used in natural mating or artificial insemination.