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Effects of Method of Egg Yolk Addition and of Glycerol Equilibration Time upon Post-Thawing Motility and Metabolic Activity of Frozen Bull Semen1
Abstract
SUI~ARY A semen sample from each of six Holstein and Guernsey bulls was used in a study to deternfine the effects of method of egg yolk addition to diluents and the glycerol equilibration time prior to freezing upon the quality of frozen semen stored for 2 wk, three months, and six months at --79 C. Criteria for the quality of the thawed semen were the per cent of motile sperm, utilization of total reducing sugars, and lactic acid production. By all three semen quality criteria, there was some advantage in adding egg yolk to the glycerol as well as to the nonglycerol portions of the diluent, although the effect was not statis- tically sig'nificant. Six-, 12-, and 18-hr glycerol equilibration periods were superior (P < 0.01) to a 2-hr equilibration period by all semen quality criteria. No interaction terms were of special significance. The large decrease in the number of motile spermatozoa of the bull which occurs during the usual in vitro freezing procedures empha- sizes the need for better semen freezing pro- cedures. The original procedure for adding egg yolk to semen diluents was through the nmdium of the initial nonglycerol portion of the two-phase diluent system (16). Subsequently, tIafs and Elliott (7) experinlentally studied this pro- cedure. Both the post-thawing motility and the fertility of the semen were highest with the procedure in which egg yolk was present in both the initial and the glycerol-containing di- Iuents, in contrast to having the egg yolk pres- ent in the initial diluent only. Polge and Row- son (16) originally suggested that, with an egg yolk diluent, sperm survival was best with a glycerol equilibration period of from 15 to 20 hr. Other workers (2, 12), using egg yolk diluents, found that a 6-hr glycerol equilibra- tion period was adequate for optimum sper- matozoa survival. Saroff and ~Iixner (18), using 2-, 6-, 12-,
... Concurrently, it also has relevance for semen collection and processing, due to time needed prior to cryopreservation [17]. Studies carried out since the late 1960s [18] and more recently [19] reported the beneficial effects of equilibration before cryopreserving bovine sperm. Although equilibration of ram semen for 24 h did not improve fertility [15], the value of an intermediate equilibration interval (12 h) for cryopreserving ram sperm has apparently not been reported. ...
The objective was to evaluate the effects of various antioxidants and duration of pre-freezing equilibration on cryopreservation of ram semen. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control), or supplemented with reduced glutathione (GSH: 0.5, 1.0, and 2.0 mM), superoxide dismutase (SOD: 5, 10, and 20 U/mL), or catalase (CAT: 5, 10, and 20 U/mL), and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 12 or 24 h after equilibration. Total antioxidant capacity was determined in the in natura extenders and after addition of semen samples for various durations of processing (fresh/dilute, throughout refrigeration, and post-thaw). Plasma membrane (PI-CFDA), acrosome integrity (FITC-PNA), and mitochondrial membrane potential (JC-1) were determined in fresh/diluted, refrigerated, and post-thaw samples. Post-thaw sperm motility was assessed with a computerized analysis system (CASA). There were no significant differences in acrosome damage or mitochondrial membrane potential after refrigeration and freeze-thaw, regardless of antioxidant addition. Sperm plasma membrane integrity was worse (P < 0.05) with cryopreservation immediately after equilibration (average 20.1 ± 8.3; mean ± SD) than after 12 h of equilibration (average 42.5 ± 10.9); however, the addition of SOD and CAT (10 and 20 U/mL) resulted in no significant difference between post-equilibration intervals of 0 and 12 h. Total antioxidant activity was not different (P > 0.05) among treatments after sperm addition or throughout the refrigeration and post-thaw. In conclusion, adding GSH, SOD or CAT did not increase the total antioxidant capacity of semen, nor did it enhance the quality of the post-thaw sperm. However, maintenance of ram semen at 5 °C for 12 h prior to cryopreservation reduced membrane damage of frozen-thawed sperm.
he aim of this study was to determine the effects of different pre-freezing equilibration times (2, 4 and 6 h) on quality parameters if post-thawed buck semen using soybean lecithin supplemented extender. In this study, semen samples were collected from four Mahabadi bucks. After collection, semen samples were primarily evaluated and pooled together. Afterward, pooled semen samples were divided into three equal parts and diluted in semen extender containing soybean lecithin. Then, after spending different equilibration times diluted semen samples were frozen. After thawing, sperm motility characteristics, plasma membrane integrity, and functionality, abnormality and lipid peroxidation were evaluated. Base on the obtained results showed that six h-equilibration times resulted in higher progressive motility (25.6 ± 1.39 %) compared to other groups (P<0.05). the other sperm quality parameters did not alter by different equilibration times. In conclusion, spending six h equilibration time improved only sperm progressive motility. Also, doing other professional evaluations and fertility are recommended.
The cryopreservation process cause stress physical and chemical to the spermatozoa, causing biochemistry alteration, irreversible reduction of the spermatic motility, increase of the DNA degeneration and intrcellular enzyme and lipids release. The aim of this study was to evaluate the influence of non-breeding and breeding seasons, glycerol and ethylene glycol, cryopreservation and thawing processes on stallion spermatozoa DNA-protein complex. It was compared fresh semen, diluted semen frozen without cryoprotectants, diluted semen exposured to cryoprotectants but not frozen and d) diluted semen frozen with cryoprotectants. Six stallions had 12 semen collections each. DNA-protein complex pathology was assessed by optical microscopy (1000x) using spermatozoa treated with ethanol-acetic acid 3:1 (v/v), HCl 4N at room temperature and toluidin blue 0,025% in McIlavaine buffer. Results showed that DNA-protein complex were different between frozen and not frozen spermatozoa groups (P<0,05). Frozen semen without cryoprotectants had no increasing of DNA-protein complex pathology compared to semen cryopreserved with cryoprotectant, but both showed increasing in relation to fresh and diluted semen exposured to cryoprotectants. The influence of non breeding and breeding season showed significant difference (P<0,05) in the fresh semen and fresh semen frozen without cryoprotectants. Cryopreservation process had negative influence on spermatozoa DNA-protein complex.
The influence of percent egg yolk during cooling from 37 to 5 C on ability of sperm to withstand subsequent freezing was studied with semen from each of eight bulls. Treatments were arranged factorially and included .5, 1, 2, 4, 8, 16, and 32% egg yolk, cooling times of .5, 1, 2, and 4 h, and freezing in ampules and straws. Percent egg yolk did not influence motility or percent intact acrosomes when semen was frozen in ampules. For sperm in straws, both post-thaw percentages of motile sperm and intact acrosomes were similar for sperm cooled with .5, 1, 2, 4, or 8% egg yolk and greater than with 16 or 32% egg yolk. Optimal egg yolk during cooling was not dependent upon rate of cooling, regardless of how semen was packaged for freezing. The influence of percent egg yolk during freezing was investigated in a second study. Treatments included eight bulls; 1, 2, 4, 8, 16, and 32% egg yolk; freezing times from +5 to -130 C of 3.5, 20, and 40 min; and freezing in ampules or straws. Optimal egg yolk during freezing in either ampules or straws was independent of the rate of freezing. For sperm frozen in ampules, differences in motility or the percent intact acrosomes due to egg yolk were small. However, the use of only 1 or 2% egg yolk depressed motility whereas 16 or 32% egg yolk decreased the percentage of intact acrosomes. Thus, extremely high or low egg yolk should be avoided. For sperm in straws, survival was optimal when the extended semen contained 4 to 32% egg yolk during freezing.
In a factorial design experiment with three levels of egg yolk (18.4, 23.0, and 27.6%), three levels of glycerol (6, 8, and 10%), and four glycerol equilibration times (2, 6, 12, and 18 hours) there was a progressive increase in sperm survival in all diluter treatments after freezing to −75° C. as the glycerol equilibration time was increased from 2 to 18 hours. A significant interaction between egg yolk and glycerol levels also was demonstrated. There were no significant interactions involving glycerol equilibration time and the other variables.
In a second factorial design experiment, four egg yolk levels (15, 20, 25, and 30%), five glycerol levels (5, 6, 7, 8, and 9%), and three storage times at 5° C. (0, 1, and 3 days) after thawing were used. A highly significant interaction of egg yolk and glycerol levels was again demonstrated. As the egg yolk level was increased, the glycerol level had to be increased also for optimal sperm survival after freezing. A diluter containing 20% egg yolk and 7% glycerol gave best sperm survival after freezing.
IN 1949, Polge, Smith and Parkes1 reported that glycerol had remarkable properties in protecting spermatozoa against the effects of low temperatures. The most promising results were obtained with fowl spermatozoa, which, frozen rapidly to -79° C. (solid carbon dioxide) or -192° C. (liquid air) in 15 per cent glycerol, resumed full motility on thawing. Later, it was found that fowl spermatozoa so treated were capable of effecting fertilization of the egg2. Mammalian spermatozoa have proved more refractory. The spermatozoa of several species-rabbit, guinea-pig and horse3, bull and goat4-can be revived to show some degree of motility after freezing at -79° C. in 15 per cent glycerol. However, repeated experiments with the rabbit, the only laboratory animal suitable for artificial insemination, have, as yet, resulted in the fertilization of only one egg by spermatozoa so treated3, and early tests with frozen bull spermatozoa carried out at Shinfield were unpromising, though one calf was reported to have been produced5.
There is some difference of opinion among experimenters as to the sig- nificance of the length of time that sperm cells remain in a glycerolated extender prior to freezing. The basis of this study was a comparison of results of using 0.5-hour and 18-hour equilibration times on bovine semen in egg yolk-sodium citrate and skimmilk semen extenders. The results were measured in terms of the percentage of motile sperm after thawing as compared with the percentage observed prior to freezing. The study made possible a determination of the differences between individual bulls in relation to recovery percentage and the significance of the length of storage period in evaluating semen samples. Editor. The time of the glycerol equilibration period is a matter of controversy among workers in the field of freezing bovine semen. The equilibration period may be defined as the time that sperm cells remain in a glycerolated extender prior to freezing. Polge and Rowson (4) recommended an equilibration time ranging between 15 and 20 hours when using an egg yolk extender. Miller and VanDemark (2), comparing 2-, 6-, and 18-hour equilibration times when using an egg yolk ex- tender, found the 6-hour period to be superior in terms of percentage of motile sperm cells surviving after freezing. Cragle et al. (1), also using an egg yolk extender, found 14.9 hours to be the most favorable equilibration time between 4 and 28 hours. Saroff and Mixner (5) showed a progressive increase in sperm cell survival in an egg yolk extender as the equilibration time was increased from 2 to 18 hours. O'Dell and Almquist (3), using a heated skimmilk extender, did not observe any significant difference ill sperm cell survival after freezing when using 0.5-, 4-, and 18-hour equilibration times. The studies reported here compare the effects of 0.5-hour and 18-hour equili- bration times on the freezing of bovine spermatozoa in egg yolk-sodium citrate and skimmilk semen extenders.
A series of experiments was conducted in order to detect and identify prod- ucts of glucose catabolism by bovine spermatozoa. Semen for the trials was obtained with the artificial vagina from mature Holstein bulls. The experimental procedure was begun within 2 hours after the collection of semen samples. Pooled ejaculates from three to four bulls were used in each trial; individual ejaculates included in the pool had an initial motility of at least 50% and a concentration of no less than 109 spermatozoa per milliliter. The spermatozoa were washed twice to remove seminal plasma (6) and suspended in calcium-free Ringer-phos- phate solution, pH 7.4; 2.5 × 109 total spermatozoa were placed in each incuba- tion flask. The incubation flasks were similar to those described by Bloom et al. (3). Two-tenths ml. of 10 N sulfuric acid was added to each control flask to inactivate the spermatozoa. Five thousand units of sodium penicillin was added to each flask to prevent bacterial interference, although bacterial growth should not affect results until after 3 hours at 37 ° C. (9). l~inger-phosphate solution was added in amounts such that the final volume in each flask after the addition of glucose-C ~ would be 5 ml. Flasks were gassed with nitrogen and equilibrated to 37 ° C. in a water bath. Uniformly labeled glucose-C ~ was dissolved in Ringer- phosphate solution and 0.5 ml. containing 8.5 to 8.7 ~c. was added to each flask. Flasks were incubated under nitrogen at 37 ° C.
The inclusion of 25% egg yolk in both the nonglycerol and the glycerol fractions of extenders for frozen semen was compared with including 50% egg yolk in the nonglycerol fraction and none in the glycerol fraction. The final compositions of these two extenders were identical. The average percentages of motile sperm immediately after thawing were 30.5 and 22.3 for the two methods, respectively, and the average 60- to 90-day nonreturn percentages for 1,515 first services were 68.0 and 59.7, respectively. These differences were highly significant statistically (P < 0.01).
Fructose, glucose, or xylose was added to give a final concentration of 1.0% by adding the sugar entirely in the nonglycerol fraction, equally in the nonglycerol and glycerol fractions, or entirely in the glycerol fraction. The three methods of incorporating the sugars resulted in 21.6, 28.4, and 28.4% motile sperm, respectively, immediately after thawing. The average percentages of motile sperm in the extenders containing the three sugars were: fructose, 27.9; glucose, 26.4; and xylose, 24.2. In both eases differences were highly significant (P < 0.01) when tested by means of the analysis of variance.
Experiments were designed to determine the optimal combination for per cent sodium citrate, per cent glycerol (by volume), and equilibration hours which would allow maximum survival of motile spermatozoa after being frozen for 5 days by use of dry ice. A sodium citrate–yolk glycerol diluent was used which contained approximately 24% yolk in the final dilution. A dilution rate of 20 million spermatozoa per milliliter of diluted semen was used. A three-dimensional central composite experimental design was used, which is adapted to estimating the optimal level of the factors studied. The estimated optima were: sodium citrate, 2.9%; glycerol, 7.6%; equilibration time, 14.9 hours.
As equilibration time was decreased, per cent survival decreased only slowly. An interaction between sodium citrate and glycerol was noted. Although semen from bulls was different, the pattern of response to treatments was the same with only absolute magnitude of response varying. An eosin-aniline blue live-dead stain was not satisfactory for estimating live spermatozoa when used on freshly thawed semen samples.
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