• Korean Journal of Poultry Science
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• v.48 no.4
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• pp.185-191
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• 2021

Chicken spermatozoa have the ability to survive in low-temperature environments; however, the effects of low temperature on sperm motility and acrosome damage have not been studied in detail. The present study investigated semen longevity following dilution of rooster semen with Beltsville Poultry Semen Extender (BPSE) and Lake extender in preservation vessels (1.5 mL e-tube and 0.5 mL straw). Spermatozoa motility in the closed-type vessel (0.5 mL straw) was higher than that in the 1.5 mL e-tube on day 3 of preservation (68.6±3.1% vs. 22.1±5.7%). The motility of rooster semen diluted with BPSE in 0.5 mL straw was also higher than that of the Lake extender on day 3 of preservation (57.7±5.6% vs. 37.7±5.4%). Furthermore, acrosome intactness was higher in 0.5 mL straw than in the 1.5 mL e-tube, and the rate of acrosome cap damage increased with preservation days. The present study demonstrates that a closed 0.5-mL straw vessel could be used for low-temperature semen preservation, with an increased motility rate and acrosome integrity in fresh rooster semen.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.203-207
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• 2011

The objective of this study was to determine the effect of semen extenders on the motility, viability and fertility in vitro of spermatozoa during storage of fresh boar semen diluted in different commercial extenders used for pig artificial insemination (AI). In this experiment, semen were diluted in Androhep plus, Beltsville Thawing Solution (BTS), Modena, Seminark and Vitasem LD. Five ejaculates were collected from three Duroc boars and sub-samples were diluted ($30{\times}10^6$ spermatozoa/ml) in different extenders. Semen was stored at $170^{\circ}C$ for 10 days. Sperm motility and viability was assessed using Computer-Assisted Semen Analysis (CASA) and flow-cytometry on 1, 3, 5 and 10 day post collection The motility of spermatozoa stored in different extenders was gradually decreased by increasing the duration of storage of semen. However, there was not significant1y different in the sperm motility and viability among other extenders. On the other hand, the in vitro-matured oocytes were fertilized and cultured in vitro to assess the fertility of boar spermatozoa stored for 3 and 10 days in different extenders. The percentage of morula and blastocyst were taken as indicators of fertility in vitro of spermatozoa. Therefore, there were no differences in the rate of embryos developed to the molular and blastocyst stage. There were no differences in the motility and fertility in vitro among 5 kinds of commercial boar semen extenders.

• Reproductive and Developmental Biology
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• v.40 no.1
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• pp.1-5
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• 2016

This study investigated the effects of L-carnitine (LC) and nicotinic acid (NA) on sperm viability during liquid storage at $18^{\circ}C$ in miniature pigs. $10{\mu}M$ LC and 30 mM NA, combined LC and NA (LN) were treated in fresh semen for 3, 7, and 10 days. In results, sperm survival increased in NA- and LN-treated semen on 7 and 10 days (p<0.05), mitochondrial integrity of live sperm increased in LN-treated semen on 7 days (p<0.05), but not NA-treated semen. In addition, we examined the acrosome reaction of sperm in miniature pigs. LC and NA did not influence on acrosome reaction of boar sperm. In conclusion, LC and NA effectively maintained the viability and quality of sperm during long-term storage in miniature pigs, suggesting that the combined LN may be useful for improving the semen extender for long-term liquid storage in pigs.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.5
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• pp.633-636
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• 2002

Six adult Garole rams maintained under a semi-intensive system were used as semen donors for this study. Semen was collectied daily during the monsoon season with the help of an artificial vagina and examined for its quality characteristics. Ejaculates of thick consistency, rapid wave motion, ${\geq}80%$ motility and intense movement of motile spermatozoa were diluted at the rate of 1:1 with egg yolk McIllvaine glucose diluent at $30^{\circ}C$ in water bath. Estrus in ewes was detected by parading aproned rams of proven vigour at 12 h intervals. The ewes (54 Malpura and 23 Avikalin) in estrus were artificially inseminated with fresh diluted ram semen. The overall conception rate was 94.8%, (range 91.7 to 100%). The overall lambing percent was 80.5 with a range of 75.0 to 84.6%. There was no significant (p>0.05) difference in lambing and conception rate because of individual rams. Fertility was significantly lower (p<0.05) in ewes of less than two years and more than six years of age. Breed (Malpura and Avikalin) effect was not observed in conception and lambing rate (p>0.05). No significant difference (p>0.05) in birth weight and 12 month weight was observed between Garole${\times}$Avikalin and Garole${\times}$Malpura crossbred lambs but there was significant (p<0.05) difference at three month and six months body weight of both the crossbred lambs.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.261-265
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• 2004

The present study was conducted to assess sperm characteristics in miniature-pig. The semen samples were transported to the laboratory at 17℃ within 3 hours after collection. The extended semen was stored at 17℃, and sperm quality was evaluated at 0, 1, 3, 5 and 7 days after storage. The semen volume of miniature-pig (62±22㎖) was significantly (p<0.05) lower than that of Duroc (155±25㎖) and Yorkshire (154±23㎖). Significant differences were also observed in sperm concentrations. During 3 days of storage, sperm viability did not differ among miniature-pig, Duroc and Yorkshire. However, the viability was significantly (p<0.05) lower in miniature-pig than in Duroc and Yorkshire semen after Day 3 of storage. In abnormality, acrosome intactness and intensity, there were no differences among miniature-pig, Duroc and Yorkshire semen. On the other hand, the viability of frozen-thawed sperm in miniature-pig was significantly (p<0.05) lower than in that of Duroc and Yorkshire. This study also examined CTC patterns in frozen-thawed spermatozoa. The rates of AR pattern were higher in miniature-pig than in Duroc and Yorkshire. However, no difference was found in F, B and AR patterns. The results of present study suggest that further research is necessary to develop of semen extender and freezing methods to improve sperm quality in miniature-pig.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.207-212
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• 2009

The aim of present experiment was to examine commercial synthetic extender(AndroMed) for semen cryopreservation of Korean Black Bull. Semen was collected from a Korean Black Bull using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by AndroMed. The pellect was diluted to final sperm concentration of $5{\times}10^5/ml$ by doubling in every 10 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 1 hr at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 minutes and above 10 cm for 10 min. And then the frozen straw was plunged to $LN_2$. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. Hanwoo semen was used as KPN (Korea Proven Bull Number) in this experiment. The survival rates was significantly higher in fresh semen than frozen semen ($80{\pm}14%\;and\;43{\pm}11%$). However, the motility rates was similar (80.7% and 66.4%). The survival and motility rates were higher in 5cm, 10 min treatment group than the other two groups in straw-located height and duration above $LN_2$ ($50{\pm}14%$ and 70.7% vs, 33.18% and $65{\pm}7%$ vs, 30.14% and 65.7%, respectively). The development rates to cleavage was higher in Black Cow than Hanwoo semen (62.2%, 64.4%), However, The development rates to blastocyst was higher in Hanwoo than Black cow semen (25.9%, 23.0%). In conclusion. The present results that acceptable fertilization and cryopreservation could be obtained by in vitro fertilization with frozen-thawed semen using a synthetic semen extender (AndroMed).

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.267-272
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• 2010

The present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen in our laboratory for in vitro fertilization (IVF) experiments due to lack of fresh semen. Antibiotics were added in the frozen semen extender (kanamycin and gentamicin) and in vitro culture (IVC) medium (gentamicin) for further inhibiting growth of microorganisms. Nevertheless, proliferations of microorganisms were observed in IVC culture drop during culturing of IVF embryos using frozen semen. Randomly 3 samples were taken from the liquid semen, frozen semen and egg yolk. Contaminated IVC medium, frozen-thawed semen, liquid semen and egg yolk were cultured in de Man, Rogosa and Sharpe (MRS) agar medium. Whitish colonies were detected in contaminated IVC drop, frozen-thawed semen samples and egg yolk but no colonies were formed in liquid semen samples. Gram-negative and rod-shaped identical bacteria were found in both frozen-thawed semen sample and contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to manufacturer's instruction with identification value (% ID) 94.3% and T index 0.88. Antibiotic susceptibility tests were done according to Clinical and Laboratory Standards Institute (CLSI) by using ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin test. Among them Enterobacter cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, kanamycin but susceptible to tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin. From these findings it could be suggested that this contamination sources might be from egg yolk.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.71-74
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• 2006

Hypoosmotic swelling test (HOST) is used for evaluating the plasma membrane function and fertilizing ability in mammal spermatozoa. However, HOS solutions and experimental conditions have not been determined clearly for assessing canine spermatozoa. This study was conducted to examine the HOS solutions and assay conditions, including incubation time (30 to 120 min), storage temperature (4, 17 and $20^{\circ}C$), semen status (fresh and frozen). Maximum spermatozoal plasma membrane swelling was obtained in an 150 mOsm Na-citrate/Fructose solutions with an incubation time for 45 min. The storage temperature and semen status affected the percentage of HOS positive spermatozoa. The HOS test adapted to canine spermatozoa in this study was simple and highly consistent assay with good repeatability. The optimal condition of HOST in canine spermatozoa is an 150 mOsm Na-citrate/Fructose solutions with an incubation time for 45 min regardless of semen storage temperature and semen status.

• Korean Journal of Poultry Science
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• v.42 no.2
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• pp.109-116
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• 2015

Ogye rooster sperm chromatin status can be detected using well established sperm assays. In this paper, a simple and fast method to monitor rooster sperm chromatin status could be employed in field for assessment of chicken sperm quality. Using standard bright field microscope, Diff-Quik stains can be reproducibly, easily and routinely monitored with simple staining. The presence of abnormal chromatin staining of rooster sperm was determined by darker stain in head. In the fresh semen, the viabilities of three tested Ogye spermatozoa were 93.53%, 82.42% and 90.63% and normal chromatin rates were 87.96%, 74.25% and 85.10% respectively. However, after freezing, the rates of viability of thawed semen were reduced to 69.58%, 61.98% and 72.20% and normal chromatin rate also reduced to 58.91%, 48.49% and 63.34%. A significant correlation between live sperm and normal sperm nuclei was 0.875 in fresh semen and 0.513 in frozen semen. After incubation of sperm at $37^{\circ}C$ for 5min, the rates of viability, chromatin normality and sperm head activity were shown as $90.63{\pm}1.28%$, $82.44{\pm}8.09%$ and $66.68{\pm}10.29%$ in fresh semen. However, the rates of thawed semen were reduced to $67.92{\pm}7.55%$, $56.92{\pm}12.15%$ and 47.32{\pm}5.02%, respectively. The relationship between chromatin normality and sperm head movements in fresh and thawed semen were 0.564 and 0.540, respectively. With these results, the chicken sperm normality could be assessed by the Diff-Quik staining that could be used for chromatin status of sperm head and activated morphology of live spermatozoa, as a simple and rapid staining method.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.494-498
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• 2008

MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) reduction assay is a method that validates the viability of an active cell. Dehydrogenase in mitochondria converts yellow colored insoluble tetrazolium salt to purple colored water-soluble formazan. Sperm also have mitochondria in the midpiece, therefore sperm viability could be evaluated by MTT reduction assay. Several studies have already demonstrated the capability of application of the MTT reduction assay to sperm of several species in Hepes-BSA buffer. Because most liquid semen was diluted in extender like BTS (Beltsville Thawing Solution), Modena or Androhep when it is used or transferred, semen needed another dilution in Hepes-BSA buffer to assess sperm viability. In this study, we evaluated boar sperm viability especially in BTS extended semen and compared the efficiency of this test with eosin-nigrosin staining. We used the fresh BTS extended semen from a local A.I center. Semen sample was diluted to $3.0{\times}10^7$ sperms/ml in BTS. The rates of formazan production were measured in 96-well microtiter plates immediately and 1h after incubation at $17^{\circ}C$ using a spectrophotometer at wave length 560 nm. Simultaneously, split samples of the same semen were tested, using eosin-nigrosin staining to compare the efficiency of the MTT assay of sperm viability in BTS. The correlation between the results of these tests was calculated using Student-t test and ANOVA. The results revealed a strong correlation between the results of MTT reduction rate and the results that were simultaneously determined by eosin-nigrosin staining at 1 h. In conclusion, the MTT reduction test was an effective and simple method to validate sperm viability and it could be used as a simple tool to evaluate sperm viability in the local A.I center and laboratory.