| We always use egg-yolk extender to cryopreserve buffalo semen, but the protect effect of frozen semen could be worse and the use ratio of buffalo far less than cattle. So it is great significant that we research and develop the formula of cryopreservation extender of buffalo semen. The effect of cryopreservation can be improved, good breeding bull can be made full use of, the buffalo species can be accelerated improved. Although extenders contained animal origin (egg-yolk) have well protective effect for semen cryopreservation, they have been phase-out in the international trade because of possibility of easy to spread disease. In order to develop new buffalo semen extender of none animal origin and improve the effect of semen cryopreservation. This study was based on semen from four Betelnut-jiang buffalos. In order to discuss the effects of soybean lecithin replaces egg-yolk and adding different density of melatonins or the new antibiotics (GTLS), and the effect of incorporating soybean lecithin with melatonin or GTLS, which based on egg-yolk extender. We evaluate the motion characteristics of sperm, motility rate and abnormal rate of sperm, which compare to the egg-yolk extender regarded as the control group. GTLS is composed of gentamincin (G)、tylosin (T)、 lincomycin (L) and spectinomycin (S). We wanted to screen out appropriate extenders for the buffalo. Results are as follows.一、Improving the formula of egg-yolk extender.1. In the test of the effects of adding melatonin into the egg-yolk extender:We set up three density of melatonins. They were low (10-5mol/L)、middle(10-4mol/L) and high (10-3mol/L) density. Results show that, the motion characteristics、motility rate of frozen semen were improved and abnormal rate were reduced. The cryopreservation effect was droped while adding overtoped density of melatonin. The VCL (14.26μm/s) and VSL (5.09μm/s) of low density group had improved13.44%(P<0.05) and16.48%(P<0.05) compared to control group. The VSL (3.73μm/s) and motility rate (26.20%) of high density group had reduced14.65%(P<0.05) and22.53%(P<0.05) compared to control group. Middle group got the best results. Motility rate had improved (P>0.05) and abnormal rate had reduced (P>0.05).2. In the test of the effects of new antibiotics (GTLS) replacing penicillin and streptomycin (SP):We divided three densities of GTLS which were high (x10), middle (x1) and low (x0.1) density. The middle density of GTLS(x1) were500μg/mL、 100μg/mL、300μg/mL%600μg/mL. We used three different density of GTLS replaced the SP separately. Results show that, the quality of frozen semen improved as the increased density of antibiotics, but overtoped can be effect the quality. VSL (5.35μm/s), LIN (39.84%), WOB (59.62%) and STR (66.43%) were improved a lot compared to the control group (P<0.05) under the middle density of GTLS. Compared to the control group, motility rate of middle (40.58%) and low density (42.61%) group had improved and abnormal rate of high density group (33.02%) had reduced (P>0.05). In conclusion, we can acquire the best results when replacing the SP of Middle density of GTLS.二、The formula research of none animal origin extender1. In the test of the effects of soybean lecithin replacing egg-yolk:We set three density of soybean lecithin, which are3%,4%and5%. Results show that, the VCL (15.88μm/s), VSL (6.06μm/s) and VAP (9.19μm/s) was higher than the control group (12.82μm/s、4.57μm/s and7.72μm/s) under3%soybean lecithin (P<0.05). The motility rate (29.05%) of frozen-thawed was lower than the control group (36.45%) under5%soybean lecithin (P<0.05). The abnormal rate of all treatment groups (3%、4%、5%) were no significant difference compared to the control group (38.55%、35.47%、29.05%)(P>0.05). In conclusion, both3%and4%soybean lecithin were a good choice, especially for3%soybean lecithin.2. In the test of the effects of soybean lecithin combine with new antibiotics:We combine soybean lecithin (3%、4%、5%) with GTLS (x0.1、x1、x10) each other and add them into egg-yolk extender. We found that, the motion characteristics and motility rate of frozen semen were getting better and abnormal rate was getting worse as the increased density of soybean lecithin and antibiotics. In the condition of3%,4%and5%soybean lecithin with GTLS of high density, motility rate (25.07%、24.07%、22.82%) of sperm after frozen-thawing had a significant decrease compare to the control group (34.45%)(P<0.05). VCL (14.58μm/s) under3%soybean lecithin combine with GTLS of middle density got an improvement of13.73%compared to the control (12.82μm/s)(P<0.05) and motility rate (36.37%) was higher and abnormal rate (41.37%) was lower than the control (34.45%,42.25%)(P>0.05).3. In the test of the effects of soybean lecithin couple with melatonin:We combine soybean lecithin (3%、4%、5%) with melatonin (10-5mol/L、10-4mol/L、10"3mol/L) each other and add them into egg-yolk extender. We found that, the motion characteristics and motility rate were in proportion and abnormal rate was inversely proportion to the density of soybean lecithin and melatonin. VSL (4.01μm/s)、VAP (6.25Dμm/s) and motility rate (30.77%) of sperm were lower than the control group (4.89pm/s>7.50μm/s、36.88%)(P<0.05) when3%soybean lecithin combine with10-3mol/L melatonin. As a whole, this test was not ideal. The experiment explain that it is not a good choice for semen cryopreservation when soybean lecithin combine with melatonin. |
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