| Dezhou donkey is an economically important source for meat, milk and Chinese medicineEjiao production, especially in China. However, important aspects of their male reproductivetechnologies (particularly of artificial insemination) remain unknown thus far. The objective ofour study is to develop a new cryopreservational formula and optimization method, and find theinfluential factors to the frozen-thawed semen quality, by studying the skim-milk, sodium caseinand caseinate in Dezhou donkey semen freezing process protection, antioxidant protection ofSOD and CAT in freezing medium of egg-yolk, influence of factors,such as seasonal, sexualbehavior, body measurements, on semen quality after frozen-thawed semen, so as to lay afoundation for Dezhou donkey chilled preservation semen.1. Effect of skim-milk, casein and caseinate on cooled preserved Dezhou donkey spermatozoaIn the donkey species, the application of cooled semen artificial insemination could aid thesurvival of endangered breeds. A suitable method for the cryopreservation of donkey semenrequires an optimal cryopreservation protocols and an optimal extender and it would be veryvaluable for the management of genetic diversity of the endangered Dezhou donkeys. Ourpreliminary work indicated that a particular medium (Tris-Citric acid-Glucose containing20%hen egg yolk and7%glycerol, pH=7.1) and procedure (filtering, semen dilution, centrifugation,resuspension，cooling and freezing velocity) may be effective for cryopreservation of Dezhoujackass spermatozoa as evaluated by sperm motility, mitochondria activity, acrosome integrity,DNA integrity, membrane integrity and pregnancy rate after AI with frozen-thawed semen.Casein, caseinate, and skim-milk on cooled preserved Dezhou donkey spermatozoa canguarantee the stability of the diluent for batch production by reduce the biological sourcecomposition in the diluent, and low glycerin concentration also can reduce the toxicity ofdilution of sperm. The purpose of this study is to explore the effect of skim-milk, casein andcaseinate in semen cryopreservation under the condition of low glycerin content (2.5%).Experimental results shows that with base dissolving casein and caseinate, when theconcentration of casein and caseinate up to1.2%, obvious protective effect was observed, theaverage survival rate is40.6%. There is no significant difference (P＞0.05) when continue toincrease the concentration of casein. On this basis, using casein diluent (1.2%or1.4%casein and1%sodium caseinate) mixed with skim-milk according to1:1, average living rate is better thancasein and caseinate after frozen. When the final concentration of casein is0.7%, the average living rate is43.6%, but no significant difference (P>0.05). After frozen, compared with theresults of egg-yolk diluents, the average survival rate of casein diluent is low (40.6%casein VSyolk: VS43.9%). Subsequent studies have illustrated that spermatozoa processed by thisprocedure are63.9%conception rate by insemination to194jennies and also may have moreacrosomal integrity and plasma membrane integrity.The results showed the casein diluent wasmore suitable for cryopreservation semen production.2. Effect of SOD and CAT in sperm cryopreservation of Dezhou donkeyThere appear to be two major classes of sperm damage: oxidative and osmotic. Oxidativedamage is mainly caused by oxidative stress, and oxidative stress is caused by the imbalance ofreactive oxygen species (ROS) and antioxidant. Cryopreservation exposes spermatozoa tostressful conditions, leading to reduced cell viability. In bovine semen, a decrease in antioxidantactivity following cryopreservation has been reported. Furthermore, feeeze-thawing of equineand bovine spermatozoa has been associated with an increase in ROS generation. However, littleis known about the dynamics of enzymatic or non-enzymatic antioxidant defense systems duringcryopreservation of donkey semen and the influence of semen extenders. The main purpose ofthis research is to study the effect of SOD (superoxide dismutase) and CAT (catalase) in thesemen freezing process. Super oxygen anion (O2-) and hydrogen peroxide (H2O2) is the mostcommon factors associated with the potential oxidative damage of sperm. O2-can be convertedto H2O2by SOD, and then the H2O2can be translated into H2O and O2which is harmless tosperm cells by CAT. Thereby we can eliminating ROS damage. In this experiment, we measuredthe activity of SOD, CAT, GSH-Px (Glutathione peroxidase), T-AOC in raw semen, semendiluent, diluted semen and frozen-thawed semen. In raw semen, three kinds of enzyme activity issignificantly higher than the semen diluent, the diluented semen and the frozen-thawed semen (P＜0.01), there is no significant difference between the diluented semen and frozen-thawed semen(P＞0.05). There is no significant difference of T-AOC activity in raw semen, the diluentedsemen and frozen-thawed semen, but the T-AOC activity is low in semen diluent. Adding SODand CAT in semen diluent can improve frozen-thawed sperm survival rate,membrane integrityand mitochondrial activity. The optimal concentration of SOD and CAT is300and400U/mL,respectively.3. The correlation between the season, body meaurements, testicular parameters sexualbehavior and semen production in Dezhou donkey.There is some correlation between bull’s body image traits and reproductive characteristic. Wecan select good bulls through bull’s body image marker, but there is no report if there wasrelationship between donkey’s body image character and reproduction at home and abroad. Purpose of this paper:①Correlation between season and frozen-thawed semen, in order todeterminate the feasibility of semen collection and semen freezing in the non-rutting season;②The relationship between season and sexual behavior of jack, to set-up the theory basis forsemen collection;③Correlation analysis between body weight, body measurements andsurvival rate, volume, concentration, total sperm count, which provide theoretical guidance forthe kind of jack breeding;④The relationship between survival rate and sexual behavior, in orderto provide theroretical guidance for jack semen collection.⑤Correlation analysis betweensemen volume and density, find the most suitable frequency of ejaculations. Results show that:①There is no significant difference between estrus-off season and estrus season about survivalrate in four of five donkeys（P＞0.05）.②Compared with estrus-off season and rutting seasonof all five donkeys, there is no difference in FLEH and COP (P＞0.05), there is a significantdifference in WME and EL (P＜0.05).③There is significant correlation between body heightand ejaculatory amount (R=0.423，P＜0.05), significant correlation between body length（R=0.502）, scrotum circumference（R=0.501）, circumference of cannon bone（R=0.717）,tajiri long（R=0.748）and total sperm count（P＜0.05，P＜0.01).④There is no correlationbetween WME, FLEH, EL, COP and surivival rate of frozen-thawed semen (R＜0.4, P＞0.05).⑤It is a negatively correlation between volume of ejaculation and semen concentration.All theabove results suggest that excellent jack must meet the following factors: size up to fit, testiclesdevelop good. There is no relationship between season and survival rate of frozen-thawed semen,indicates that semen production can conduct throughout the whole year, but it need to set-up jackin rutting season such as winter. The right frequency of semen collection is every other day ortake three times a week. |
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