Nghiên cứu ảnh hưởng của tỷ lệ pha loãng, chất bảo quản, chất chống đông và quy trình làm lạnh đến chất lượng tinh trùng cá mú cọp epinephelus fuscoguttatus (forsskal, 1775) bảo quản trong nitơ lỏ

B GIO DC V O TO TRNG I HC NHA TRANG -o0o - PHM VN DIN NGHIấN CU NH HNG CA T L PHA LONG, CHT BO QUN, CHT CHNG ễNG V QUY TRèNH LM LNH N CHT LNG TINH TRNG C M CP Epinephelus fuscoguttatus (FORSSKAL, 1775) BO QUN TRONG NIT LNG LUN VN THC S KHNH HềA - 2017 B GIO DC V O TO TRNG I HC NHA TRANG -o0o - PHM VN DIN NGHIấN CU NH HNG CA T L PHA LONG, CHT BO QUN, CHT CHNG ễNG V QUY TRèNH LM LNH N CHT LNG TINH TRNG C M CP Epinephelus fuscoguttatus (FORSSKAL, 1775) BO QUN TRONG NIT LNG LUN VN THC S Ngnh: Nuụi trng thy sn Mó s: 60620301 Quyt nh giao ti: 90/Q-HNT ngy 04/02/2016 Quyt nh thnh lp H: 232/Q-HNT ngy 24/02/2017 Ngy bo v: 23/3/2017 Ngi hng dn khoa hc: TS Lấ MINH HONG Ch tch hi ng: PGS.TS PHM QUC HNG Khoa sau i hc: HONG H GIANG KHNH HềA 2017 LI CAM OAN Tụi xin cam oan, kt qu nghiờn cu ca ti: Nghiờn cu nh hng ca t l pha loóng, cht bo qun, cht chng ụng v quy trỡnh lm lnh n cht lng tinh trựng cỏ mỳ cp Epinephelus fuscoguttatus (Forsskal, 1775) bo qun nit lng ti ny l mt phn ni dung ca d ỏn c tớnh lý húa hc v bo qun lnh tinh trựng cỏ mỳ cp ti Vit Nam Qu Quc t cho khoa hc (IFS) Thy in ti tr vi ch nhim d ỏn l TS Lờ Minh Hong cú mó s A/5165-1 Tỏc gi lun Phm Vn Din iii LI CM N Trc tiờn, tụi xin trõn trng cm n Ban lónh o Chi cc Qun lý cht lng Nụng lõm Sn v Thy sn thuc S Nụng nghip v Phỏt trin nụng thụn tnh C Mau, ni tụi cụng tỏc ó to iu kin cho tụi tham gia khúa hc o to thc s chuyờn ngnh Nuụi trng thy sn khúa 2014 2016 ti Trng i Hc Nha Trang Tụi xin trõn trng cm n Ban giỏm hiu trng i hc Nha Trang, Khoa Sau i hc, cỏc quý thy cụ giỏo Vin Nuụi Trng Thy Sn ó ging dy v giỳp tụi sut thi gian hc v ó ng ý sp xp cho tụi thc hin ti ny Tụi xin by t lũng cm n sõu sc n TS Lờ Minh Hong, ngi thy ó tn tỡnh giỳp tụi quỏ trỡnh lm ti v hon thin lun ny Tụi xin by t lũng bit n sõu sc n TS Nguyn Th Thanh Thựy - Vin nghiờn cu Nuụi trng Thy sn ó cung cp ngun cỏ b m cú cht lng tt sut thi gian thc hin ti Tụi cng xin cm n cỏc bn th lp CHNT 2014-3 ó luụn quan tõm giỳp tụi sut quỏ trỡnh hc v nghiờn cu; xin cm n phũng thớ nghim NORAD ca Vin Nuụi Trng Thy Sn ó to iu kin v c s vt cht, trang thit b tụi hon thnh ti ny Li cm n sõu sc nht tụi xin dnh gi n nhng ngi thõn gia ỡnh, c bit l v v ó luụn ng viờn, khớch l tụi hc v nghiờn cu tt nht Nha Trang, thỏng nm 2016 Tỏc gi lun Phm Vn Din iv MC LC LI CAM OAN iii LI CM N iv MC LC v DANH MC CC T VIT TT viii DANH MC CC BNG ix DANH MC CC HèNH, TH x TRCH YU LUN VN xi M U CHNG TNG QUAN 1.1 Mt s c im sinh hc ca cỏ mỳ cp 1.1.1 H thng phõn loi 1.1.2 c im hỡnh thỏi 1.1.3 Sinh thỏi v phõn b 1.1.4 c im dinh dng 1.1.5 c im sinh trng 1.1.6 c im sinh sn 1.2 Mt s c im ca tinh trựng cỏ 1.2.1 Quỏ trỡnh to tinh 1.2.2 Cu to tinh trựng 1.2.3 c im sinh lý hc ca tinh trựng 1.2.4 Cỏc yu t nh hng n tinh trựng cỏ 1.2.4.1 Yu t lý hc ca tinh dch 1.2.4.2 Cỏc yu t húa sinh ca dch tng 10 1.2.5 Cỏc yu t nh hng n quỏ trỡnh bo qun tinh trựng cỏ nit lng 13 1.2.5.1 K thut chn cỏ thu mu 13 1.2.5.2 T l pha loóng 13 v 1.2.5.3 Cht bo qun 13 1.2.5.4 Cht chng ụng v nng cht chng ụng 15 1.2.5.5 Quy trỡnh lm lnh 17 1.2.5.6 Phng phỏp ró ụng 18 1.3 Tỡnh hỡnh nghiờn cu bo qun tinh trựng nit lng 18 1.3.1 Trờn th gii 18 1.3.2 Vit Nam 21 CHNG VT LIU V PHNG PHP NGHIấN CU 25 2.1 a im, thi gian v i tng nghiờn cu 25 2.2.1 a im 25 2.1.2 Thi gian nghiờn cu 25 2.1.3 i tng nghiờn cu 25 2.2 S ni dung nghiờn cu 25 2.3 Chn cỏ c v thu tinh 25 2.3.1 Chn cỏ c 25 2.3.2 Vut v thu tinh 26 2.4 ỏnh giỏ mt v hot lc tinh trựng trc thớ nghim 26 2.4.1 Xỏc nh mt 26 2.4.2 Xỏc nh hot lc ca tinh trựng 26 2.5 Quy trỡnh bo qun tinh trựng cỏ nit lng 27 2.6 B trớ thớ nghim 28 2.6.1 Thớ nghim 1: nh hng ca cht bo qun (DMSO:10%) n hot lc v tc tinh trựng cỏ mỳ cp 28 2.6.2 Thớ nghim 2: nh hng ca cht chng ụng n hot lc v tc tinh trựng cỏ mỳ cp 29 2.6.3 Thớ nghim 3: nh hng ca t l pha loóng 29 2.6.4 Thớ nghim 4: nh hng ca th tớch bo qun n hot lc v tc tinh trựng cỏ mỳ cp 30 vi 2.6.5 Thớ nghim 5: nh hng ca quy trỡnh lm lnh n hot lc v tc tinh trựng cỏ mỳ cp 30 2.5 Phng phỏp x lý s liu 31 CHNG KT QU NGHIấN CU V THO LUN 32 3.1 nh hng ca cht bo qun n hot lc v tc tinh trựng cỏ mỳ cp 32 3.2 nh hng ca cht chng ụng n hot lc v tc tinh trựng cỏ mỳ cp 33 3.3 nh hng ca t l pha loóng n hot lc v tc tinh trựng cỏ mỳ cp 35 3.4 nh hng ca th tớch bo qun lờn hot lc v tc tinh trựng cỏ mỳ cp 36 3.5 nh hng ca phng phỏp lm lnh lờn hot lc v tc tinh trựng cỏ mỳ cp 38 CHNG KT LUN V XUT í KIN 40 4.1 Kt lun 40 4.2 xut ý kin 40 TI LIU THAM KHO 41 vii DANH MC CC T VIT TT ASP: Artificial semina plasma MPRS: Marine Ringer Solution àm: micromet g: gam s: giõy LG-ASP2: Longtooth grouper Artificial seminal plasma ES1-3: Epinephelus septemfasciatus EL3: Epinephelus lanceolatus ELRS3: Epinephelus lanceolatus ringer solution FM1: Quy trỡnh lm lnh bc FM2: Quy trỡnh lm lnh bc FM3: Quy trỡnh lm lnh bc viii DANH MC CC BNG Bng 1.1 Cỏc cht bo qun dựng cho bo qun lnh tinh trựng mt vi loi cỏ 15 Bng 1.2 Cỏc loi cht chng ụng dựng nghiờn cu bo qun lnh tinh trựng 15 Bng 1.3 Cỏc loi cht chng ụng dựng cho bo qun lnh ca mt vi loi cỏ 16 Bng 1.4 Kt qu bo qun lnh tinh trựng mt s i tng cỏ bin nit lng 23 Bng 2.1 Mt s c im lý hc ca tinh dch cỏ mỳ cp 27 Bng 2.2 Thnh phn cht bo qun s dng thớ nghim 28 ix DANH MC CC HèNH, TH Hỡnh 1.1 Hỡnh dng ngoi ca cỏ mỳ cp Epinephelus fuscoguttatus (Forsskal, 1775) Hỡnh 1.2 Phõn b ca cỏ mỳ cp trờn th gii Hỡnh 1.3 Cu to tinh trựng Hỡnh 2.1 S nghiờn cu bo qun tinh trựng cỏ mỳ cp nit lng 25 Hỡnh 3.1 nh hng ca t l pha loóng lờn hot lc v tc tinh trựng cỏ mỳ cp 32 Hỡnh 3.2 nh hng ca cht bo qun lờn hot lc v tc tinh trựng cỏ mỳ cp 34 Hỡnh 3.3 nh hng ca cht chng ụng lờn hot lc v tc tinh trựng cỏ mỳ cp 35 Hỡnh 3.4 nh hng ca th tớch bo qun lờn hot lc v tc tinh trựng cỏ mỳ cp 37 Hỡnh 3.5 nh hng ca quy trỡnh lm lnh lờn hot lc v tc tinh trựng cỏ mỳ cp 38 x 62 Horvỏth, A and Urbanyi, B (2000) The effect of cryoprectants on the motility and fertilizing capacity of cryopreserved African catfish Clarias gariepinus (Burchell 1822) sperm Aquac Res 63 Horvỏth, ., Urbỏnyi, B., and Mims, S.D (2009) Cryopreservation of sperm from 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2.04803 58.9439 68.3894 57.00 76.00 1:3 83.6667 3.46410 1.15470 81.0039 86.3294 76.00 87.00 1:6 64.3333 6.24500 2.08167 59.5330 69.1337 55.00 75.00 1:9 51.2222 5.26255 1.75418 47.1771 55.2674 41.00 57.00 36 65.7222 12.84770 2.14128 61.3752 70.0693 41.00 87.00 1:1 1.1733E2 10.13657 3.37886 109.5417 125.1250 102.00 140.00 Di1:3 1.3589E2 5.27836 1.75945 131.8316 139.9462 125.00 143.00 Di1:6 1.2444E2 12.17694 4.05898 115.0844 133.8045 110.00 139.00 Di1:9 1.0744E2 10.07610 3.35870 99.6993 115.1896 90.00 120.00 36 1.2128E2 14.05827 2.34304 116.5211 126.0344 90.00 143.00 Total D_VT Mean Std Deviation Total Hot lc Duncan Subset for alpha = 0.05 T l pha loóng N 1:9 51.2222 1:1 63.6667 1:6 64.3333 1:3 Sig 83.6667 1.000 795 1.000 Vn tc Duncan Subset for alpha = 0.05 T l pha loóng N 1:9 1.0744E2 1:1 1.1733E2 1:6 1.2444E2 1:3 Sig 1.3589E2 1.000 132 1.000 nh hng ca cht bo qun n hot lc v tc tinh trựng Descriptives 95% Confidence Interval for Mean N E_MOT ELRS3 Mean Std Deviation Std Error Lower Bound Upper Bound Minimum Maximum 31.8889 6.75360 2.25120 26.6976 37.0802 20.00 40.00 ELS3 56.3333 6.61438 2.20479 51.2491 61.4176 45.00 64.00 ES1-3 71.5556 5.57026 1.85675 67.2739 75.8372 65.00 80.00 LG-ASP2 53.7778 9.94708 3.31569 46.1318 61.4238 40.00 66.00 150 mM NaCl 40.7778 9.32440 3.10813 33.6104 47.9451 20.00 54.00 300 mM Glucose 46.1111 7.21880 2.40627 40.5622 51.6600 35.00 54.00 DGS1 43.8889 10.10500 3.36833 36.1215 51.6563 30.00 54.00 DGS2 49.8889 9.96800 3.32267 42.2268 57.5510 40.00 66.00 MPRS 34.8889 5.75423 1.91808 30.4658 39.3120 25.00 40.00 81 47.6790 13.79477 1.53275 44.6287 50.7293 20.00 80.00 Total E_VAP ELRS3 ELS3 ES1-3 1.0444 E2 8.63295 2.87765 97.8086 111.0803 85.00 112.00 1.1133 12.94218 E2 4.31406 101.3851 121.2816 90.00 125.00 1.1878 E2 9.92192 3.30731 111.1511 126.4044 95.00 130.00 1.0956 E2 8.03292 2.67764 103.3809 115.7302 92.00 117.00 LG-ASP2 150 mM NaCl 300 mM Glucose DGS1 DGS2 MPRS Total 1.0533 10.52378 E2 3.50793 97.2440 113.4226 90.00 120.00 1.0578 13.06501 E2 4.35500 95.7351 115.8204 90.00 120.00 1.0467 12.09339 E2 4.03113 95.3709 113.9625 90.00 118.00 1.0622 E2 9.01080 3.00360 99.2959 113.1485 92.00 116.00 1.0456 E2 7.66667 2.55556 98.6624 110.4487 92.00 114.00 1.0785 10.84218 E2 1.20469 105.4544 110.2493 85.00 130.00 81 Hot lc Duncan Subset for alpha = 0.05 Cht bo qun N ELRS3 31.8889 MPRS 34.8889 150 mM NaCl DGS1 43.8889 43.8889 300 mM Glucose 46.1111 46.1111 46.1111 DGS2 49.8889 49.8889 49.8889 LG-ASP2 53.7778 53.7778 ELS3 56.3333 ES1-3 34.8889 40.7778 40.7778 71.5556 Sig .436 128 193 143 Vn tc Duncan Subset for alpha = 0.05 EXTENDER ELRS3 N 104.4444 061 116 1.000 MPRS 104.5556 DGS1 104.6667 150 mM NaCl 105.3333 300 mM Glucose 105.7778 DGS2 106.2222 LG-ASP2 109.5556 109.5556 ELS3 111.3333 111.3333 ES1-3 Sig 118.7778 239 079 nh hng ca cht chng ụng n hot lc v tc tinh trựng Descriptives N C_MOT DMA5% Std Deviatio Mean n 95% Confidence Interval for Mean Std Error Lower Bound Upper Bound Minimum Maximum 43.3333 7.81025 2.60342 37.3298 49.3368 35.00 53.00 DMA10% 64.8889 6.56591 2.18864 59.8419 69.9359 55.00 78.00 DMA15% 69.8889 3.78961 1.26320 66.9759 72.8018 63.00 75.00 DMA20% 56.2222 8.07431 2.69144 50.0158 62.4287 50.00 70.00 DMSO5% 62.4444 5.36449 1.78816 58.3209 66.5680 50.00 67.00 DMSO10% 70.4444 7.65034 2.55011 64.5639 76.3250 55.00 80.00 DMSO15% 82.5556 6.76593 2.25531 77.3548 87.7563 67.00 88.00 DMSO20% 20.2222 4.14662 1.38221 17.0348 23.4096 15.00 25.00 Me5% 27.3333 4.30116 1.43372 24.0272 30.6395 18.00 32.00 Me10% 20.3333 3.80789 1.26930 17.4063 23.2603 15.00 25.00 Me15% 11.7778 2.72845 90948 9.6805 13.8751 7.00 15.00 Me20% 8.4444 2.69774 89925 6.3708 10.5181 5.00 12.00 EG5% 54.3333 6.02080 2.00693 49.7053 58.9613 50.00 65.00 EG10% 66.7778 7.37865 2.45955 61.1060 72.4495 55.00 78.00 EG15% 69.5556 3.39526 1.13175 66.9457 72.1654 63.00 75.00 EG20% 51.7778 2.27913 75971 50.0259 53.5297 50.00 55.00 G5% 20.6667 4.71699 1.57233 17.0409 24.2925 13.00 25.00 G10% 47.2222 11.69164 3.89721 38.2352 56.2092 20.00 58.00 G15% 20.7778 4.63081 1.54360 17.2182 24.3373 12.00 26.00 G20% 12.4444 5.29413 1.76471 8.3750 16.5139 5.00 23.00 180 44.0722 24.08482 1.79518 40.5298 47.6147 5.00 88.00 1.1544 8.53099 2.84366 108.8869 122.0019 E2 102.00 132.00 1.2378 10.04711 3.34904 116.0549 131.5007 E2 102.00 133.00 1.2744 6.83943 2.27981 122.1872 132.7017 E2 114.00 135.00 1.1122 6.97814 2.32605 105.8583 116.5861 E2 99.00 120.00 1.2011 15.56795 5.18932 108.1445 132.0777 E2 92.00 130.00 1.1711 4.34294 1.44765 113.7728 120.4494 E2 110.00 125.00 1.3689 3.51584 1.17195 134.1864 139.5914 E2 133.00 143.00 1.0444 4.92725 1.64242 100.6570 108.2319 E2 94.00 110.00 Me5% 99.6667 4.84768 1.61589 95.9404 103.3929 91.00 104.00 Me10% 91.4444 4.36208 1.45403 88.0914 94.7974 85.00 97.00 Me15% 78.1111 5.81903 1.93968 73.6382 82.5840 65.00 82.00 Me20% 68.8889 6.00925 2.00308 64.2698 73.5080 55.00 75.00 1.1889 7.91272 2.63757 112.8066 124.9711 E2 106.00 132.00 1.2167 12.52996 4.17665 112.0353 131.2980 E2 93.00 133.00 1.2744 6.83943 2.27981 122.1872 132.7017 E2 114.00 135.00 1.1233 6.46142 2.15381 107.3666 117.3000 E2 99.00 120.00 91.2222 5.16667 1.72222 87.2508 95.1937 80.00 97.00 1.1089 6.84552 2.28184 105.6270 116.1508 E2 102.00 120.00 85.1111 6.56591 2.18864 80.0641 90.1581 75.00 95.00 Total C_VAP DMA5% DMA10% DMA15% DMA20% DMSO5% DMSO10% DMSO15% DMSO20% EG5% EG10% EG15% EG20% G5% G10% G15% G20% Total 78.4444 7.40120 2.46707 72.7554 84.1335 60.00 84.00 1.0703 19.97692 1.48899 104.0895 109.9660 E2 55.00 143.00 180 Hot lc Duncan CRYOPRO TECT Subset for alpha = 0.05 N Me20% 8.4444 Me15% 11.7778 G20% 12.4444 DMSO20% 20.2222 Me10% 20.3333 G5% 20.6667 G15% 20.7778 Me5% DMA5% 43.3333 G10% 47.2222 47.2222 EG20% 51.7778 51.7778 EG5% 54.3333 DMA20% 56.2222 DMSO5% 62.4444 DMA10% 64.8889 64.8889 EG10% 66.7778 66.7778 EG15% 69.5556 DMA15% 69.8889 DMSO10% 70.4444 DMSO15% Sig 27.3333 82.5556 178 859 1.000 164 104 134 144 077 1.000 Vn tc Duncan Subset for alpha = 0.05 CRYOPROTE CT N 10 11 Me20% 68.8889 Me15% 78.1111 G20% 78.4444 G15% 85.1111 G5% 91.2222 Me10% 91.4444 Me5% DMSO20% G10% 1.1089E2 1.1089E2 DMA20% 1.1122E2 1.1122E2 EG20% 1.1233E2 1.1233E2 DMA5% 1.1544E2 1.1544E2 1.1544E2 DMSO10% 1.1711E2 1.1711E2 1.1711E2 1.1711E2 EG5% 1.1889E2 1.1889E2 1.1889E2 1.1889E2 DMSO5% 1.2011E2 1.2011E2 1.2011E2 1.2011E2 EG10% 1.2167E2 1.2167E2 1.2167E2 DMA10% 1.2378E2 1.2378E2 DMA15% 1.2744E2 EG15% 1.2744E2 DMSO15% Sig 85.1111 99.6667 1.0444E2 1.0444E2 1.3689E2 1.000 066 097 185 075 051 054 126 100 069 nh hng ca th tớch pha loóng n hot lc v tc tinh trựng Descriptives N V_HL 0.25 ml Mean Std Deviatio n 95% Confidence Interval for Mean Std Error 83.1111 6.39227 2.13076 Lower Bound 78.1976 Upper Bound 88.0246 Minimum Maximum 70.00 90.00 1.000 0.50 ml 72.2222 9.05232 3.01744 65.2640 79.1804 65.00 90.00 1.50 ml 79.2222 8.89913 2.96638 72.3817 86.0627 70.00 90.00 27 78.1852 9.12325 1.75577 74.5761 81.7942 65.00 90.00 0.25 ml 1.3611E2 9.96800 3.32267 128.4490 143.7732 118.00 150.00 0.50 ml 1.1456E2 13.08731 4.36244 104.4958 124.6154 95.00 143.00 1.50 ml 1.0889E2 7.16667 2.38889 103.3801 114.3977 103.00 123.00 27 1.1985E2 15.55314 2.99320 113.6992 126.0045 95.00 150.00 Total V_VT Total Hot lc Duncan Subset for alpha = 0.05 TH TCH N 0.50 ml 72.2222 1.50 ml 79.2222 0.25 ml 79.2222 83.1111 Sig .083 325 Vn tc Duncan Subset for alpha = 0.05 TH TCH N 1.50 ml 108.8889 0.50 ml 114.5556 0.25 ml Sig 136.1111 257 1.000 nh hng ca phng phỏp lm lnh n hot lc v tc tinh trựng Descriptives 95% Confidence Interval for Mean N F_HL F_VT Mean Std Deviation Std Error Lower Bound Upper Bound Minimum Maximum FM1 40.1111 5.57773 1.85924 35.8237 44.3985 35.00 50.00 FM2 83.4444 5.10174 1.70058 79.5229 87.3660 75.00 90.00 FM3 84.1111 4.37163 1.45721 80.7508 87.4714 75.00 90.00 Total 27 69.2222 21.53054 4.14355 60.7050 77.7394 35.00 90.00 FM1 1.0667E2 6.26498 2.08833 101.8510 111.4824 95.00 112.00 FM2 1.3211E2 10.04296 3.34765 124.3914 139.8308 118.00 146.00 FM3 1.3656E2 10.12560 3.37520 128.7723 144.3388 118.00 146.00 Total 27 1.2511E2 15.95989 3.07148 118.7976 131.4246 95.00 146.00 Hot lc Duncan Phng phỏp lm lnh Subset for alpha = 0.05 N FM1 40.1111 FM2 83.4444 FM3 84.1111 Sig 1.000 781 Vn tc Duncan Phng phỏp lm lnh Subset for alpha = 0.05 N FM1 106.6667 FM2 132.1111 FM3 136.5556 Sig 1.000 305 ... bc T KHểA Tinh dch, tinh trựng, cht bo qun, cht chng ụng, th tớch bo qun, cỏ mỳ cp Epinephelus fuscoguttatus, hot lc tinh trựng xii M U Cỏ mỳ cp Epinephelus fuscoguttatus (Forsskal, 1775) l loi... VN DIN NGHIấN CU NH HNG CA T L PHA LONG, CHT BO QUN, CHT CHNG ễNG V QUY TRèNH LM LNH N CHT LNG TINH TRNG C M CP Epinephelus fuscoguttatus (FORSSKAL, 1775) BO QUN TRONG NIT LNG LUN VN THC S Ngnh:... cu ca ti: Nghiờn cu nh hng ca t l pha loóng, cht bo qun, cht chng ụng v quy trỡnh lm lnh n cht lng tinh trựng cỏ mỳ cp Epinephelus fuscoguttatus (Forsskal, 1775) bo qun nit lng ti ny l mt phn