• Journal of Embryo Transfer
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• v.33 no.2
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• pp.75-84
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• 2018

The cryopreservation has been extensively applied in many cells including spermatozoa (semen) during past several decades. Especially, the canine spermatozoa cryopreservation has contributed on generation of progeny of rare/genetically valuable dog breeds, genome resource banking and transportation of male germplasm at a distant place. However, severe and irreversible damages to the spermatozoa during cryopreservation procedures such as the thermal shock (cold shock), formation of intracellular ice crystals, osmotic shock, stress of cryoprotectants and generator of reactive oxygen species (ROS) have been addressed. According as a number of researches have been conducted to overcome these problems and to advance cryopreservation technique, several analytical methods have been employed to evaluate the quality of the fresh or cryopreserved canine spermatozoa in regards to the motility, morphology, integrity of membrane and DNA, mitochondrial activity, ROS generation, binding affinity to oocytes, in vitro fertilization potential and fertility potential by artificial insemination. Because the study designs with certain application of analytical methods are selective and varied depending on each experimental objective and laboratory condition, it is necessary to establish the normal reference data of the fresh or cryopreserved canine spermatozoa for each analytical method to monitor experimental procedure, to translate raw data and to discuss results. Here, we reviewed the recent articles to introduce various analytical methods for the canine spermatozoa as well as to establish the normal reference data for each analytical method in the fresh or cryopreserved canine spermatozoa, based on the results of the previous articles. We hope that this review contributes to the advancement of cryobiology in canine spermatozoa.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.385-390
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• 2011

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in ${\alpha}$ 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. After thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

• Korean Journal of Poultry Science
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• v.44 no.1
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• pp.59-65
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• 2017

This study examined factors affecting the analysis of motility of chicken semen. The viability of spermatozoa was estimated using varying dilution ratios and supplementation with BSA or fatty acid free (FAF)-BSA as protein sources in semen diluent. Fresh semen was examined after preparing dilutions in beltsvile poultry semen extender (BPSE) of 1/8, 1/16 and 1/32 at $25^{\circ}C$. The motility of incubated semen at each dilution was observed at 3 min (89.9%, 69.9% and 53.2%), 30 min (86.7%, 71.4% and 51.7%), 1 h (89.5%, 74.0% and 53.5%) and 3 h (78.5%, 66.5% and 45.7%), respectively. The addition of BSA or FAF-BSA to BPSE diluent significantly increased the viability of semen in 1/32 dilution with results of 53.2% (control), 84.8% (BSA) and 92.9% (FAF-BSA) (p<0.05). This phenomenon was also observed in the dilution of frozen semen, where FAF-BSA treatment increased the viability of thawed semen from 17.6% to 34.0% in a 1/8 dilution (p<0.05). When the protein sources were used in the dilution, the survival rates of diluted chicken semen were also increased with time lapse. These results show that FAF-BSA may act to protect chicken semen and is suitable as a basic component of chicken semen diluent for the method of analyzing rooster semen after freezing.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.219-222
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• 2011

Eight female Himalayan tahrs (Hemitragus jemlahicus) were estrus-synchronized, and transcervically inseminated with frozen-thawed semen in September, 2009, about 2 to 3 months earlier than their natural breeding season. Intravaginal progesterone-releasing devices were inserted into vaginas of six Himalayan tahrs on September 7, and the other two on September 8 to suppress luteal function of ovaries. The devices had been placed deep inside the vagina prior to withdrawal on September 23. A day before CIDR removal, a combination of PMSG 400 IU and hCG 200 IU was intramuscularly injected. Forty hours later, frozen-thawed semen was transcervically inseminated. Pregnancy diagnosis was performed 39 days later by analyzing progesterone level of serum. Every treatment was done under anesthesia inducted by xylazine injection. In conclusion, vaginal discharge of cervical mucus, hormonal changes induced by implant-typed or muscularly injectable hormones and widening of cervix enough to insert an insemination gun into uterine body were achieved in non-breeding season. Moreover, the first inseminated Himalayan tahr, 36 hours after CIDR removal was assumed to be pregnant but the fetus may have been lost due to the use of anesthetic drug.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.5
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• pp.622-637
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• 2017

Fatty acids such as n-3 and n-6 polyunsaturated fatty acids (PUFA) are critical nutrients, used to improve male reproductive performance through modification of fatty acid profile and maintenance of sperm membrane integrity, especially under cold shock or cryopreservation condition. Also, PUFA provide the precursors for prostaglandin synthesis and can modulate the expression patterns of many key enzymes involved in both prostaglandin and steroid metabolism. Many studies carried out on diets supplemented with PUFA have demonstrated their capability to sustain sperm motility, viability and fertility during chilling and freezing as well as improving testis development and spermatogenesis in a variety of livestock species. In addition to the type and quantity of dietary fatty acids, ways of addition of PUFA to diet or semen extender is very crucial as it has different effects on semen quality in male ruminants. Limitation of PUFA added to ruminant ration is due to biohydrogenation by rumen microorganisms, which causes conversion of unsaturated fatty acids to saturated fatty acids, leading to loss of PUFA quantity. Thus, many strategies for protecting PUFA from biohydrogenation in rumen have been developed over the years. This paper reviews four aspects of PUFA in light of previous research including rumen metabolism, biological roles, influence on reproduction, and strategies to use in male ruminants.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.163-169
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• 2014

The objective of this study was to evaluate the characteristics of Korean Native Cattle sperm frozen-thawed with L-cysteine and/or catalase. The semen from bulls was collected by the artificial vagina method, and Triladyl containing 20% egg-yolk and/or L-cysteine (L), catalase (C) and L-cysteine + catalase was added to the diluted semen for cryopreservation. The results showed that sperm viability was significantly higher in the L-cysteine + catalase ($69.49{\pm}3.16%$) group than in the control ($60.5{\pm}3.94%$) group (p<0.05). Acrosome damage was significantly lower in the L-cysteine ($17.12{\pm}1.08%$) group than in the control ($21.46{\pm}1.14%$), catalase ($20.54{\pm}0.76%$), and L-cysteine + catalase ($19.29{\pm}0.65%$) groups (p<0.05). In addition, the level of intact mitochondria in the spermatozoa was significantly higher in the L-cysteine ($58.65{\pm}1.39%$) group than in the control ($50.63{\pm}2.37%$) group (p<0.05). The hydrogen peroxide level in the frozen-thawed sperm was significantly lower in the L-cysteine ($3.74{\pm}1.66%$), catalase ($4.65{\pm}1.87%$), and L-cysteine + catalase ($8.11{\pm}2.15%$) groups than in the control ($13.22{\pm}1.6%$) group (p<0.05). The glutathione level was significantly higher in the L-cysteine ($1.33{\pm}0.03%$) group than in the control ($1.08{\pm}0.06%$), catalase ($1.05{\pm}0.02%$) and L-cysteine + catalase ($1.11{\pm}0.03%$) groups (p<0.05). In conclusion, L-cysteine and catalase could protect the membrane of Korean Native Cattle sperm from damage during sperm cryopreservation. Especially, L-cysteine was more effective for keeping acrosomes and mitochondria intactness during sperm cryopreservation.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.10
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• pp.1276-1281
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• 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.

• Journal of Embryo Transfer
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• v.23 no.2
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• pp.115-118
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• 2008

Glycerol is the cryoprotectant most frequently used to freeze semen in several of species. The objective of the present study was to compare the effect of three different glycerol concentrations (4, 6 or 8%, v/v) on frozen-thawed dog sperm survival rate. Ejaculates from 9 dogs collected by digital manipulation were pooled and assessed by macroscopic and microscopic criteria. Semen was divided into 3 aliquots, which were centrifuged and the sperm pellets rediluted with first Tris-glucose-citric acid extender. After 1 h cooling at $4^{\circ}C$, second extender containing 4, 6 or 8% glycerol was added, respectively. The semen was loaded into 0.25 ml straws and frozen and stored in liquid nitrogen and thawed. Sperm vigor, live:dead spermatozoa ratio using HOS test, and sperm morphology using $Spermac^{(R)}$ stain were evaluated. After thawing, there were no significant differences among groups in vigor, viability and morphology. In conclusion, the three glycerol concentrations (4, 6 or 8%) can be used successfully in cryopreservation of canine semen. Therefore the use of 4% glycerol in the extender has less toxic effect and reduces of freezing injuries.

• Korean Journal of Agricultural Science
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• v.47 no.3
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• pp.657-665
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• 2020

The objective of this study was to investigate whether supplementation of pentoxifylline (PTX; phosphodiesterase inhibitor) to thawed boar semen improves the post-thaw motility of sperm and affects the efficiency of artificial insemination (AI) and further development. To determine the concentration of PTX for AI, frozen-thawed semen was incubated with 0, 5, 10, and 20 mM PTX in an extender freezing medium, respectively, after thawing. Kinematic properties of sperm were examined with a computer-assisted semen analysis (CASA) system. In addition, viability and mitochondrial activity were also tested by LIVE/DEAD and a MitoTracker kit. There were no significant differences in the kinetic parameters of thawed sperm between control and treatment groups, but overall assessment parameters such as motility and rapid progressive were higher in the 10 mM PTX group. In the viability and mitochondrial assay, there were no significant differences observed in the PTX treatment, compared to the control. For further analysis, artificial inseminations were performed using frozen semen and 10 mM PTX treated cryopreserved semen, respectively. There were no differences in pregnancy rates and fetus weights among the groups until 30 and 40 days, but litter size was reduced and relatively low-birth weight was observed in the PTX group. In summary, our findings suggest that enhancement of in vitro sperm quality or non-toxicity supplemented by PTX may have detrimental effects on fetus development.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.4
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• pp.177-182
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• 2018

Objective: To investigate the effects of in vitro myo-inositol (Myo-Ins) supplementation of cryopreserved human semen on the cryo-survival rate (CSR). Methods: Semen samples were obtained from 41 infertile men. Following routine semen analysis, each sample was divided into two equal aliquots (0.5 mL each). One aliquot was treated with 1 mg of Myo-Ins dissolved in $10{\mu}L$ of sperm preparation medium. The second aliquot was treated with $10{\mu}L$ of the same medium (control). Both aliquots were incubated for 20 minutes prior to freezing to slow the freezing process. The frozen samples were examined for post-thaw percentages of total motility (TM), progressive motility (PM), and the CSR, defined as the percentage of post-thaw TM divided by the percentage of pre-freeze TM and multiplied in 100. The results were expressed as median and interquartile range (25th and 75th percentiles). Results: The pre-freeze TM (50% [30%-50%]) and PM (35% [20%-35%]) were significantly higher than the post-thaw TM and PM in the MyoIns group (15% [10%-35%] and 10% [5%-20%]; p<0.001 and p<0.001, respectively) and the control group (10% [6%-30%] and 5% [3%-15%]; p<0.001 and p<0.001, respectively). The CSR of the 41 semen aliquots supplemented with Myo-Ins (40% [25%-70%]) was significantly higher than that of the control samples (30% [13%-58%], p=0.041). The CSR of the 26 abnormal semen samples that were supplemented with Myo-Ins (38% [20%-50%]) was significantly higher than that of the control samples (23% [12%-30%], p=0.031). Conclusion: In vitro Myo-Ins supplementation of ejaculated human sperm from infertile men resulted in a significant increase in the CSR in samples with abnormal pre-freeze sperm parameters.