Background: Using cryovial for freezing dog spermatozoa provides a practical method to increase extended sperm volume and shorten the time required for equilibration by using a simple freezing techniques. The purpose of this study was to determine the optimal thawing condition for dog sperm cryopreservation using cryovials. Methods: For sperm freezing, cryovials with 200 × 106 sperm/mL were cooled after the addition of tris egg yolk extender (TEY) at 4℃ for 20 min, then TEY with 4% glycerol was added and equilibrated for another 20 min before being aligned over LN2 vapor for another 20 min and plunged directly into LN2. Spermatozoa were thawed in a water bath at 37℃ for varying times (25 sec, 60 sec, 90 sec, and 120 sec) in the first experiment. In the second experiment, spermatozoa were thawed in a water bath at various temperatures and times (37℃ for 1 min, 37℃ for 1 min with gentle stirring, 24℃ for 24 min, and 75℃ for 20 sec). In these experiments, the effect of thawing conditions on motility parameters, viability (SYBR-14/PI), and acrosome integrity (PSA/ FITC) of spermatozoa were investigated. Results: The post-thaw sperm motility parameters, viability, and acrosome integrity were not significantly different across the experimental groups. Conclusions: In this study, the characteristics of spermatozoa frozen using cryovials were not significantly affected by various thawing conditions.

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 frozenthawed 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 freezingthawing, 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.

The present study was conducted to investigate the effect of different heights from liquid nitrogen (LN2) vapor on sperm motility and morphology after frozen-thawing. Two ejaculates were collected from 2 fertile Hanwoo bulls (A and B) by using artificial vagina at Hanwoo Research Institute. After collection, ejaculates were transferred to laboratory immediately and diluted with semen extender (Optixcell, France). Sperm dilutions were extended to a final concentration of 40 x 106 sperm/ml, and cooled at 4°C for 4 h and loaded to 0.5 ml straws. The straws were divided into 2 groups. Straws were placed in 3 or 9 cm of LN2 vapor for 14 min and then plunged into LN2 tank and cryopreserved until evaluation. Sperm motility and motility parameters (total motility, VSL with 25μm≥, VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were evaluated by sperm class analysis (SCA, IVOS, Spain) after frozen-thawed. In bull A, 3cm group showed higher percentages of total motility, VSL with 25μm and VAP compared those with 9cm group (98.0 vs. 93.4%, 62.4 vs. 54.0% and 98.6 vs. 93.2%, 3 vs. 9 cm, irrespectively; p<0.001). In bull B, frozen-thawed sperm of 3cm group showed higher percentages of VSL with 25μm, VCL, VSL, VAP and BCF compared with those of 9cm group (43.5 vs. 26.0%, 123.8 vs. 111.6 μm, 62.9 vs. 57.3 μm and 81.5 vs. 72.5 μm; 3 vs. 9 cm, irrespectively; p<0.001). The viability and acrosomal integrity of spermatozoa were evaluated by Trypanblue/Giemsa staining method divided into 4 groups; live and intact acrosome (LIA), live and damaged acrosome (LDA), dead intact acrosome integrity (DIA), dead damaged acrosome (DDA). In bull A, frozen-thawed sperm of 3 and 9cm groups showed no significant difference in LIA, LDA, DIA and DDA. In bull B, 3 cm group showed higher LIA and lower DIA compared with those of 9 cm group (73.2 vs. 23.7% and 23.7 vs. 32.2%, 3 vs. 9 cm, irrespectively; p<0.001). We suspected that 3 cm vapor on LN2 vapor might be affected positively spermatozoa viability and acrosomal integrity compared with 9 cm group. In conclusion, semen freezing procedure in the present study will improve sperm quality after frozen thawing.

Cryopreservation of germ cells from genetically proven animals could be a source of restoration tools from the risk of extinction or disappearance of wanted characteristics. Using frozen semen, the genetic gains of Korean native cattle have been increased greatly for 70 years. The preservation of genetic resources as a form of frozen semen straw has limited availability due to the numbers. To circumvent this weakness of frozen semen, we tested two re-freezing methods with different initial thawing temperatures using frozen Korean proven semen and rare breed semen from albino, black and chikso breeders. It has been known that human sperm could resist to cryo-damages by repeated freeze-thaw cycles, but not for Korean proven bulls number (KPN) or for rare breeds. Total 7 frozen semem from brindled(2), black(1), Korean Albino(2) and KPN(1) bulls were used for our research. After thawing straws under 5°C/2min or 37°C/40sec with low temperature water bath and thermo jug, spermatozoa were re-diluted with triladyl diluents after first thawing and re-frozen. Sperm motilities were compared between animals and treated groups after re-thawing. Mean values of motility and viability of refrozen/thawed sperm for expansion of the number of straws were significantly higher in 5°C than in 37°C (P< < 0.05). However, the activity of viable sperm thawed at 5°C was significantly decreased after first and second thawing. It is estimated that re-freezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa.

This study investigated the effects of 27.12 MHz radio frequency (RF) heating on heat transfer phenomena during the thawing process of frozen food. To determine the velocity of the RF thawing machine, samples were frozen at -80oC and subjected to different power treatments. The phase change times (-5 to 0oC) of frozen radish were 30, 26, 13, and 8 min; those of pork sirloin were 38, 25, 11, and 5 min; those of rump were 23, 17, 11, and 6 min; those of chicken breast were 42, 29, 13, and 9 min; and those of tuna were 25, 23, 10, and 5 min at 50, 100, 200, and 400 W, respectively. The heating limit temperatures of the radish, pork sirloin, rump, chicken breast, and tuna samples were 19.5, 9.2, 21.8, 8.8, and 16.8oC at 50 W; 23.5, 15.5, 27.3, 12.3, and 19oC at 100 W; 42, 26.9, 45.7, 22.1, and 39.4oC at 200 W; and 48.5, 54.7, 63.6, 57.3, and 44.9oC at 400 W. These results suggest that high-power RF improves thawing velocity and heating limit temperatures, and that an improvement on the operation of the RF thawing machine, according to food temperatures, is needed.

본 연구는 다양한 냉동방법(강제송풍식냉동, 극저온냉동, 일반냉동)과 해동방법(자연해동, 유수해동, 초음파해동, 전자레인지해동)을 조합하여 처리했을 때 표고버섯의 물리적 품질에 미치는 영향을 관찰하고, 표고버섯의 품질을 유지하는 가장 효과적인 냉·해동 공정을 탐색하는 것이 목적이었다. 급속냉동법(강제송풍식냉동, 극저온냉동)으로 처리한 경우 표고버섯의 해동감량, 보수력, 수분함량은 큰 변화 없이 품질을 유지 하는데 효과적인 것으로 관찰되었다. 특히 보수력은 저속냉동(일반냉동) 및 저속해동(자연해동) 처리의 유무가 영향을 미치는 것으로 나타났다. 전단력의 경우 냉동방법에 의한 영향보다, 전처리 및 해동방법에 더 영향을 받는 것을 판단된다. pH 및 색도는 냉동방법 및 해동방법에 따라 거의 차이가 없는 것으로 관찰되었다. 표고버섯의 물리적 특성을 효과적으로 유지 할 수 있는 처리 조합은 급속냉동(극저온냉동) 및 급속해동(유수해동, 초음파해동, 전자레인지해동)을 한 경우이며, 또한 유지하고 하는 물리적 특성에 따라 냉동 및 해동방법을 다양하게 조합하여 처리한다면, 식품의 품질을 보존하는데 더욱 효과적일 것으로 사료된다.

본 연구는 냉동식품 형태의 별미밥 HMR 개발을 위한기초 데이터를 확립하고자 냉동 및 해동 조건에 따른 취반미의 물리화학적 특성의 변화를 비교하였다. 본 실험에서는 냉동방법으로 강제송풍방식과 저온에서 자연적으로 냉동하는 방식을 사용하였으며, 냉동 취반미는 전자레인지의출력세기를 200, 600 및 1000W로 조절하여 시료의 중심부 온도가 100oC에 도달할 때까지 해동하였다. 분석결과 냉동속도는 일반냉동을 하는 것보다 강제송풍식 냉동을 하는 것이 약 300배 이상 빨랐으며, 해동은 동결 속도가 빠르고 전자레인지 출력이 셀수록 시간이 단축되었다. 냉해동한 취반미의 수분함량 변화는 모든 조건에서 대조구보다낮은 함량을 나타내었으며, 특히 600W에서 해동할 경우수분의 손실이 많은 것으로 나타났다. 취반미를 냉해동 한후 색도의 변화에 있어서는 대조구보다 백색도와 적색도는감소, 황색도는 증가하는 경향을 보였으며 강제송풍식 냉동을 한 취반미가 전체적인 색 변화가 적게 나타났다. 물성의 변화는 일반냉동을 한 취반미의 경도가 높게 나타났으며, 특히 600W에서 해동한 처리구가 높게 나타났다. 점착성의 경우에는 대조구보다 모든 처리구에서 감소하는 경향을 보였으나 조건에 따른 유의적 차이는 나타나지 않았다. 강제송풍식 냉동을 한 취반미의 미세구조는 냉해동 과정에서 밥알의 중심부에 균열이 발생하였고, 1,000W에서해동한 처리구에서 중심부에 비교적 큰 기공이 형성되었으며 600W에서 해동한 처리구의 경우에는 표면의 두께가가장 두껍게 나타났다. 냉해동한 취반미의 물리화학적 변화를 전반적으로 보았을 때 냉동 속도가 빠르고 해동시 전자레인지의 출력이 1,000W일 때 대조구와 비교하여 가장변화가 적은 것으로 나타났다.

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℃) 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℃ and 37℃) 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℃ 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℃ 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.

본 연구는 0.5 ml straw를 이용한 정자 동결융해 시 융해 온도가 동결정자의 성상에 미치는 영향을 파악하고 미니 돼지의 동결정액에 최적화된 적정 융해 조건을 찾기 위하여 실시하였다. 정액동결 straw를 37, 50 및 70℃에서 각각 5초, 10초 및 45초간 융해하여 생존율(SYBR-14/PI staining), 정자원형질막기능검사 (Hypoosmotic Swelling Test) 및 첨체반응율 (CTC : chlorotetracycline staining)을 검사 한 결과 70℃에서 5초간 융해한 정자의 생존율과 CTC 결과가 37℃와 50℃에서 10초 및 45초간 융해한 처리구보다 유의적(p<0.05)으로 높은 생존율과 낮은 비율의 첨체 반응율을 얻었다. 따라서 미니 돼지의 동결 정액은 고온에서 단시간 융해를 하는 것이 정자의 성상에 유리한 것으로 사료된다.

This study was conducted to investigate the changes in quality characteristics of frozen maesil according to thawing methods. The quality of maesil thawed in microwave oven was superior to those thaw in refrigerating temperature(5 ˚) and in room temperature(25 ˚). Drip loss of maesil thawed in microwave oven was 3.2±0.2%. The total content of free sugars of maesil was 426.6 mg%, and 3% of them was decreased during thawing in microwave oven. The total content of organic acids was 5,297.2 mg%, and 2.5% of them was decreased during thawing in microwave oven. The total content of free amino acids was 281.4 mg%, and 2.1% of them was decreased during thawing in microwave oven. The principle ingredients of frozen maesil was stand for the lost contents of free sugar and a content loss of free organic acid and free amino acid were the fewest by thawing. Antioxidant effect for soybean oil and linoleic acid of maesil extract were expressd POV and TBA values. Antioxidative activity of fresh maesil extract was highest followed by maesil thawed in microwave oven, thawed in refreezing temperature (5˚)and room temperature (25˚)

본 실험은 실험견의 정액 동결 시 희석 액에 첨가되는 Glycerol 농도, 동결속도, 동결보존액 첨가후 평형시간, 융해온도와 시간에 따라 정자의 생존성과 운동성을 조사하여 최적의 동결조건을 확립하기 위해 실시하였다. 1. 각기 다른 Glycerol 농도를 함유한 동결보존액에서 동결보존 후 융해하였을 때 4%의 Gly-cerol 농도에서 각각 68.87.4%, 73.28.3%로서 다른 군보다 유의하게 높은 생존율과 운동성 나타냈다(P<0.05). 2.