CN103126783B - Method for optimizing timely artificial insemination for pandas by aid of LH (luteinizing hormone) results - Google Patents
Method for optimizing timely artificial insemination for pandas by aid of LH (luteinizing hormone) results Download PDFInfo
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Abstract
The invention discloses a method for judging the time of optimizing timely artificial insemination for pandas by the aid of LH (luteinizing hormone) results. The method includes detecting LH peak values of female pandas and occurrence moments of the peak values of the female pandas on the basis of actual estradiol peak values of the female pandas and occurrence moments of the actual estradiol peak values of the female pandas; scheduling the individual pandas in normal ovulation to be subjected to primary artificial insemination within 14-30 hours after the occurrence moments of the LH peak values according to the occurrence moments of the LH peak values or/and the occurrence moments of the actual estradiol peak values, intervals among the occurrence moments of the LH peak values and the occurrence moments of the actual peak estradiol peak values, a condition that whether the pandas have oestrus peak behaviors or not and the like; scheduling the individual pandas in delayed ovulation to be subjected to primary artificial insemination within 30-36 hours after the occurrence moments of the actual estradiol peak values; and performing secondary artificial insemination for the individual pandas in 6-8 hours after the primary artificial insemination for the pandas. The method has the advantages that a final pregnancy rate of the pandas is increased to 66.67% from the original 50.00%, a birth rate due to natural mating reaches 65.63%, the primary artificial insemination time range is shortened to 16 hours from the original 24 hours, and major technological progress is obtained.
Description
Technical field
The present invention relates to a kind ofly for the in good time test-tube method of captive animal, particularly relate to the in good time artificial insemination method of a kind of LH of utilization result optimizing giant panda.
Background technology
Giant panda is the distinctive Rear Endangered Wildlife of China, himself fertility is on the low side, Female Giant Panda 1 year is oestrus for once, and only have 1～3 day in the oestrus, accept natural mating, its ovulation period may only have one day, and Captive Giant Panda natural mating ratio is on the low side, only has at present the giant panda natural mating of 50% left and right, there is the giant panda of half left and right to complete breeding by artificial insemination, so, determine that in good time in good time breeding of giant panda opportunity is the key technology that giant panda breeds.
The giant panda proposing at present breeding in good time or artificial insemination viewpoint are all based on estrogen E
2peak value, estrogen peak value time of occurrence proposes.It is 201210223931.9 that applicant has proposed application number on 07 02nd, 2012, denomination of invention is the in good time artificial insemination method > of < < giant panda >, the giant panda estradiol real peak of take goes out now as start time 0 hour, after the scope of 24 hours～48 hours in giant panda is carried out to artificial insemination for the first time, artificial insemination for the first time completes 6～8 and carries out artificial insemination for the second time after as a child again, final giant panda pregnancy rate has obtained and has significantly improved, with compare in the past giant panda artificial insemination pregnancy rate and bring up to 42.11% by 20.41%, turned over more than one times.
But, because giant panda ovulation also relates to other a plurality of factors such as lutropin, therefore only according to estrogen peak value time of occurrence, judge that in good time breeding time is defective.
Lutropin is a kind of of promoting sexual gland hormone, and referred to as LH, it acts on ripe ooecium, can cause and ovulates and generate corpus luteum.Research about giant panda LH at present rarely has report.
Zeng Guoqing, Jiang Guangtai, Yankee duty etc. lutropin in serum and urine, the variation of progesterone and 17 beta estradiol content. animal journal .1990.36 (1): 63-69, giant panda LH has been carried out to preliminary study, LH detects the vitro bioassay method that adopts, LH content and variation thereof in 4 inferior giant panda blood and urine have been carried out to Preliminary Determination, just to oestrus urine sample gathered once every day, and blood sample all carries out when artificial insemination anesthetized animal, result tentatively finds that giant panda occurs LH peak value in the peak period of oestrusing, LH peak value appears at after estrogen peak value.
Lin Zengyun, Zhu Zhongyong, Chen Yuchun. LH, E during giant panda oestruses and urinates the onset of ovulation
2endocrine research. Chengdu: the .1991.66-70 of Sichuan science tech publishing house, adopted different assay method (adopting iodine mark mankind lutropin hLH radioimmunoassay) to measure 2 giant panda LH, every day is collected a urine sample in the peak period of oestrusing, the result of result and Zeng Guoqing is difference to some extent, giant panda E
2oestrusing peak second day or the 3rd day, synchronously reach peak-peak with LH.
Li Xuebing, Huang Xiangming, Li Guanghan etc. the ELISA of giant panda urine hormone in oestrus measures and breeding in good time. Chengdu international Panda protection scientific seminar collection of thesis. Chengdu: the .1994:247-250 of Sichuan science tech publishing house; adopt enzymoimmunoassay to detect 69 giant panda urine in oestrus LH; gather a urina sanguinis every day; LH measures test kit and adopts U.S. Tetcan company to provide; the result comparing class of its result and Zeng Guoqing seemingly, finds that giant panda urine LH peak value appears at estrogen peak value (same day, next day or the 3rd day) after a while.
Barbara S.Durrant, Nicole Ravida, Thomas Spady et al.2006New technologies for the study of carnivore reproduction.Theriogenology, 60:1729-1736, adopt first commercialization dog serum LH to measure test kit, adopt tachysynthesis chromatograph LH algoscopy (rapid immunochromatographic LH assay) to carry out qualitative detection to LH content and variation thereof in 49 giant panda urines, find that urine LH positive test symbol appears at estrogen peak value and declines latter 24 hours.
Comprehensive above-mentioned result of study, relatively consistent result is: if using estrogen peak value as 0 day, giant panda LH appears at after estrogen peak value 0～3, but the different results of study of its exact time are different.
There is following problem in above-mentioned giant panda LH research report: first detects individual amount few (2～6), representative poor, secondly sample frequency is low, be difficult to obtain the accurate change curve of giant panda LH, because the common LH peak persistent period is shorter, if real LH peak is missed in the low meeting of sample frequency, and above-mentioned research sampling majority is every day 1 time, and Barbara is stochastical sampling; Secondly, on research method, quantitatively determination methods adopts biological assay, or people's LH measures reagent and carries out radioimmunity or enzyme-linked immunoassay, qualitative determination adopts Sanguis Canitis liquid LH to measure test kit, LH is a kind of glycoprotein hormones, the heterodimer that LH is connected to form in non-covalent bond mode by α and two glycosylation subunits of β.Have result of study to show, two glycosylation subunit homologys of the α of giant panda LH and β and Canis familiaris L. are higher, and lower with people's similarity degree.The α subunit of giant panda LH and Canis familiaris L. reach 95% and 90% at the homology of nucleotide and amino acid levels, and with people only have 84% and 74%, the β subunit of giant panda LH and Canis familiaris L. reach 92% at the homology of nucleotide and amino acid levels, and only have 84% and 75% with people, so the homology of giant panda LH and Canis familiaris L. is higher than the homology with people.Therefore theoretically, if utilize other species LH to measure test kit, detect giant panda LH, what adopt Canis familiaris L. is better than people's.But adopting at present Canis familiaris L. LH to measure test kit detection giant panda LH only only has qualitative detection research and there is no detection by quantitative research.
Wang Jianchen. < < animal genital regulating > >. Hefei: the .1998 of Anhui science tech publishing house, Zhang Jiabao. < < animal thremmatology > >. Changchun: the .1999:96 of Jilin science tech publishing house, find that LH ovulation peak plays critical effect for triggering ovulation, different animals occurs that in blood LH and the interval time of triggering ovulation are not quite similar, but be almost consistent at its LH time to peak of same animal with the interval time of triggering ovulation, therefore utilize LH index as the giant panda index of breeding in good time, for optimizing the in good time technology of artificial insemination of giant panda, be feasible theoretically.But at present without any correlational study and application report.
Summary of the invention
The technical problem to be solved in the present invention is a kind of opportunity of how utilizing LH peak value to occur, how after LH peak value or estradiol real peak occur, carry out high-efficiency artificial insemination utilize the in good time artificial insemination method of LH result optimizing giant panda.
For solving the problems of the technologies described above, a kind of in good time artificial insemination method of LH result optimizing giant panda that utilizes of the present invention, comprises the following steps:
(1) during breeding spring, described in number of patent application 201210191762.5, by time requirement, Female Giant Panda is carried out to A urine sample collection device, then to urine sample, adopt elisa to detect Female Giant Panda estradiol real peak and time of occurrence thereof, draw out the change curve of Female Giant Panda estradiol value and progesterone value;
(2) take moment of occurring Female Giant Panda estradiol real peak as start time 0 hour, adopt elisa to detect in 0 hour to 48 hours all LH values of urine samples; Using Female Giant Panda urination time as abscissa, using LH value as vertical coordinate, on the change curve of Female Giant Panda estradiol value and progesterone value, mark urination time and the corresponding determined coordinate points of LH value thereof, coordinate points is connected in turn, draw out LH value change curve, find out LH peak value and time of occurrence thereof; Described LH peak value is the maximum LH value of estradiol real peak after the moment;
(3) according to LH peak value constantly or/and estradiol real peak constantly, LH peak value determines first the time of artificial insemination jointly with estradiol real peak interval, the Female Giant Panda constantly conditions such as peak behavior that whether occur oestrusing constantly; The peak behavior of oestrusing of described Female Giant Panda refers to that Female Giant Panda occurs sticking up tail, posture to be joined, presses tail reflection, retreats phenomenons such as approaching buck, posture particularly to be joined.
Described a kind of in good time artificial insemination method of LH result optimizing giant panda that utilizes, when Female Giant Panda LH peak value be constantly less than 16 hours with estradiol real peak interval constantly and estradiol real peak constantly after 30 hours with the peak behavior that occurs oestrusing of interior Female Giant Panda, now can determine that Female Giant Panda is that normal ovulation is individual; Therefore at LH peak value, in 14～30 hours after the moment and when Female Giant Panda occurs oestrusing peak behavior, carry out first artificial insemination.
Described a kind of in good time artificial insemination method of LH result optimizing giant panda that utilizes, when Female Giant Panda LH peak value constantly with estradiol real peak constantly interval greater than 16 hours and estradiol real peak constantly after 30 hours with the peak behavior that do not occur oestrusing of interior female individuals, now can determine that Female Giant Panda is that delayed ovulation is individual; Therefore in estradiol real peak, in 30～36 hours after the moment and when Female Giant Panda occurs oestrusing peak behavior, carry out first artificial insemination.
Described any in good time artificial insemination method of LH result optimizing giant panda that utilizes carries out artificial insemination for the second time after first artificial insemination completes 6～8 hours.
Described any in good time artificial insemination method of LH result optimizing giant panda that utilizes, its artificial insemination comprises the following steps:
(1) prepare giant panda seminal fluid;
(2) anesthesia giant panda, giant panda Baoding of lying on the back, carefully cleans around giant panda vaginal orifice;
(3) will open and grow device slow circumvolve insertion giant panda vagina, by pocket lamp or other light source, observe intravaginal urethra opening and cervix uteri opening;
(4) giant panda seme-deposition device is slowly inserted to Giant Panda Uterus eck, with syringe, draw seminal fluid, be connected on seme-deposition device, seminal fluid is slowly injected in cervix uteri; Take off syringe and inject a small amount of air, so as to draining remaining seminal fluid in seme-deposition device;
(5) untie giant panda Baoding rope, allow giant panda naturally revive.
Described any in good time artificial insemination method of LH result optimizing giant panda that utilizes, its LH value adopts dog lutropin test kit to detect.
In research process, take moment of occurring giant panda estradiol real peak as start time 0 hour, adopt elisa to detect the whole urine sample LH values in 0 hour to 48 hours; Make LH change curve, using the maximum of LH as LH peak value, using there is LH peak value the urine sample excretion time as giant panda LH peak value constantly, calculate giant panda LH peak value constantly and estradiol real peak interval constantly; Adopt SPSS statistical software and descriptive data frequency analysis method to analyze the interval time (h) that 40 giant pandas urinate LH peak value and estradiol real peak oestrus, the results are shown in following table 1:
Table 1. giant panda urinates LH peak value and estradiol real peak interval time (h) oestrus:
Note: N represents sample number; A represents to exist a plurality of modes, shows minima.
As can be seen from Table 1, giant panda LH peak value and estradiol real peak range of variation interval time are larger, from 0 hour of minimum to maximum 30.22 hours, average out to 13.67 hours, wherein the individuality of great majority (60%) at 11.9 hours to 15.72 hours between approximately 12～16 hours.
Result of study shows, Female Giant Panda LH peak value constantly and the estrogen peak value time at intervals time widely different between Different Individual, between 0～30.22 hour, and be carved into during LH peak value, the interval of ovulation normally determines.Research process also finds with respect to estradiol real peak constantly, have part giant panda individuality to have delayed ovulation phenomenon first.Due to the individual LH peak value of 60% Female Giant Panda to estradiol real peak interval time in 16 hours, therefore can be according to Female Giant Panda LH peak value constantly with estradiol real peak interval constantly whether in 16 hours, tentatively judge the whether normal or delayed ovulation of Female Giant Panda; Be combined in again Female Giant Panda in after constantly 30 hours of estradiol real peak and whether occur the peak behavior of oestrusing, further determine the whether normal or delayed ovulation of Female Giant Panda.
Due to LH peak value appear at estrogen peak value after, and giant panda LH peak value time of occurrence is almost consistent with the interval time of triggering ovulation, therefore, determines that LH peak value and time of occurrence thereof are particularly important to successfully carrying out giant panda artificial insemination.
If the LH peak value of usining is constantly as reference, first artificial insemination time can be divided into early stage artificial insemination group (LH peak value constantly after 0～14 hour) and artificial insemination in late period group (LH peak value after the moment 14～30 hours), compare both farrowing rate differences, the results are shown in Table 2:
Table 2. is the impact on pregnant result with respect to LH peak value the time of artificial insemination constantly
| Breeding opportunity | Farrowing | Not farrowing | Sum | Farrowing rate % |
| Early stage |
0 | 3 | 3 | 0 |
| Artificial insemination in late period group | 5 | 1 | 6 | 83.33 |
| Add up to | 5 | 4 | 9 | 55.56 |
Adopt X 2 test to carry out significance of difference detection to early stage and artificial insemination in late period group farrowing rate, as can be seen from Table 2, early stage artificial insemination group pregnancy rate is significantly lower than group in late period, probability P=0.048<0.05, significant difference.Based on the above results, the in good time artificial insemination time of giant panda can be optimized for: suitable first the time of artificial insemination is after estrogen real peak and at LH peak value in 14～30 hours after constantly, when peak behavior appears oestrusing in giant panda, carries out.
2010～2011 Nian Jian Chengdu Panda Breeding Research Base adopt the random selection portion of the technology of the present invention to divide the giant panda that oestruses to carry out in good time artificial insemination (of the present invention group), adopting application number with 2005～2010 years is 201210223931.9, denomination of invention is that the technology (conventional group) of the in good time artificial insemination method > of < < giant panda > is compared, getting rid of health and the impact of age on result, only statistics is wherein under healthy and of the right age (6～19 years old) individual fairness doctrine, its pregnancy rate the results are shown in Table 3:
Table 3. adopts the technology of the present invention and the comparison of conventional technology of artificial insemination farrowing rate
| Artificial insemination grouping | Gestation number | Not pregnant number | Add up to | Pregnancy rate % |
| Conventional group | 7 | 7 | 14 | 50.00 |
| Of the present invention group | 4 | 2 | 6 | 66.67 |
| Add up to | 11 | 9 | 20 | 55.00 |
As can be seen from Table 3, adopt of the present invention group of technology to carry out artificial insemination to 6 healthy of the right age giant pandas, farrowing rate is up to 66.67%, reached natural mating 65.63% farrowing rate level (Feng Wen with. live in peace and to wait the different breeding wonies of .1997. on the farrow impact of effect of giant panda. Sichuan University's journal. natural science edition .34:43-47.), and higher than routine group technology of artificial insemination, than conventional technology of artificial insemination pregnancy rate, improved 16.67%.
In addition, for part Female Giant Panda, there is delayed ovulation phenomenon, should implement to postpone breeding.
Because Female Giant Panda estradiol real peak and go out to have detected exactly Female Giant Panda LH peak value as basis now and go out now is take in the present invention, then according to LH peak value constantly or/and estradiol real peak constantly, the LH peak value moment and estradiol real peak interval constantly, whether Female Giant Panda there is the conditions such as peak behavior of oestrusing, the Female Giant Panda individuality of normally ovulating is arranged within the scope of after constantly 14 hours～30 hours of LH peak value and carries out first artificial insemination, delayed ovulation Female Giant Panda individuality is arranged in after constantly 30 hours～36 hours of estradiol real peak and carries out first artificial insemination, first artificial insemination completes 6～8 and carries out artificial insemination for the second time after as a child again, final giant panda pregnancy rate brought up to present 66.67% by 50.00% of the past, reached natural mating 65.63% farrowing rate level, and by suitable first artificial insemination time scope shorten to for 24 hours present 16 hours by the past, obtained Important Techmical Progress.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 is the change curve of estradiol value, progesterone value and the LH value of the normal ovulation of the present invention typical case Female Giant Panda individuality;
Fig. 2 is the change curve of estradiol value, progesterone value and the LH value of typical delay ovulation Female Giant Panda individuality of the present invention.
In figure, identify: the change curve of 1-Female Giant Panda estradiol value, the change curve of 2-Female Giant Panda progesterone value, the change curve of 3-Female Giant Panda LH value, A-estradiol real peak coordinate points, B-LH peak coordinate point, Ec ng/mg Cr is expressed as the estradiol concentration value after creatinine is proofreaied and correct, and Pg ng/mg Cr is expressed as the Concentration of Progesterone value after creatinine is proofreaied and correct, and LH mIU/mg Cr is expressed as the LH concentration value after creatinine is proofreaied and correct.
The specific embodiment
Embodiment mono-:
The present invention is first according to described in number of patent application 201210191762.5, during breed spring, by time requirement, Female Giant Panda is carried out to A urine sample collection device, to urine sample, adopt elisa to detect Female Giant Panda estradiol real peak and time of occurrence thereof, as shown in Figure 1, draw out the change curve of Female Giant Panda estradiol value and progesterone value, obtain estradiol real peak coordinate points A.Then take moment of coordinate points A is start time 0 hour, uses the dog lutropin test kit of Bai Wo bio tech ltd, Shanghai, adopts elisa to detect in 0 hour to 48 hours all LH values of urine samples; Using Female Giant Panda urination time as abscissa, using LH value as vertical coordinate, on Fig. 1, mark urination time and the corresponding determined coordinate points of LH value thereof, coordinate points is connected in turn, draw out LH value change curve, find out LH peak value and time of occurrence thereof, obtain LH peak coordinate point B.Again, the interval that calculates coordinate points A and coordinate points B is 9.92 hours, is less than 16 hours, can tentatively determine that Female Giant Panda is that normal ovulation is individual; According to the peak behavior that occurs oestrusing of Female Giant Panda in 30 hours after coordinate points A, can further determine that Female Giant Panda is that normal ovulation is individual again.Finally, for normal ovulation Female Giant Panda individuality, in 14～30 hours after coordinate points B and when Female Giant Panda occurs oestrusing peak behavior, carry out first artificial insemination; After completing 6～8 hours, first artificial insemination carries out artificial insemination for the second time.
There is oestrusing peak behavior or/and progesterone value is carried out when in rising trend giant panda in first artificial insemination, artificial insemination is for the second time carried out when peak behavior appears oestrusing in giant panda, and twice artificial insemination all must operate according to the following steps:
A. prepare giant panda seminal fluid;
B. anaesthetize giant panda, giant panda Baoding of lying on the back, carefully cleans around giant panda vaginal orifice;
C. will open and grow device slow circumvolve insertion giant panda vagina, by pocket lamp or other light source, observe intravaginal urethra opening and cervix uteri opening;
D. giant panda seme-deposition device is slowly inserted to Giant Panda Uterus eck, with syringe, draw seminal fluid, be connected on seme-deposition device, seminal fluid is slowly injected in cervix uteri; Take off syringe and inject a small amount of air, so as to draining remaining seminal fluid in seme-deposition device;
E. untie giant panda Baoding rope, allow giant panda naturally revive.
Embodiment bis-:
The present invention is first according to described in number of patent application 201210191762.5, during breed spring, by time requirement, Female Giant Panda is carried out to A urine sample collection device, to urine sample, adopt elisa to detect Female Giant Panda estradiol real peak and time of occurrence thereof, as shown in Figure 2, draw out the change curve of Female Giant Panda estradiol value and progesterone value, obtain estradiol real peak coordinate points A.Then take moment of coordinate points A is start time 0 hour, uses the dog lutropin test kit of Bai Wo bio tech ltd, Shanghai, adopts elisa to detect in 0 hour to 48 hours all LH values of urine samples; Using Female Giant Panda urination time as abscissa, using LH value as vertical coordinate, on Fig. 2, mark urination time and the corresponding determined coordinate points of LH value thereof, coordinate points is connected in turn, draw out LH value change curve, find out LH peak value and time of occurrence thereof, obtain LH peak coordinate point B.Again, the interval that calculates coordinate points A and coordinate points B is 23.17 hours, is greater than 16 hours, can tentatively determine that Female Giant Panda is that delayed ovulation is individual; According to the peak behavior that do not occur oestrusing of Female Giant Panda in 30 hours after coordinate points A, can further determine that Female Giant Panda is that delayed ovulation is individual again.Finally, for delayed ovulation Female Giant Panda individuality, in 30～36 hours after coordinate points A and when Female Giant Panda occurs oestrusing peak behavior, carry out first artificial insemination; After completing 6～8 hours, first artificial insemination carries out artificial insemination for the second time.
There is oestrusing peak behavior or/and progesterone value is carried out when in rising trend giant panda in first artificial insemination, artificial insemination is for the second time carried out when peak behavior appears oestrusing in giant panda, and twice artificial insemination all must operate according to the following steps:
A. prepare giant panda seminal fluid;
B. anaesthetize giant panda, giant panda Baoding of lying on the back, carefully cleans around giant panda vaginal orifice;
C. will open and grow device slow circumvolve insertion giant panda vagina, by pocket lamp or other light source, observe intravaginal urethra opening and cervix uteri opening;
D. giant panda seme-deposition device is slowly inserted to Giant Panda Uterus eck, with syringe, draw seminal fluid, be connected on seme-deposition device, seminal fluid is slowly injected in cervix uteri; Take off syringe and inject a small amount of air, so as to draining remaining seminal fluid in seme-deposition device;
E. untie giant panda Baoding rope, allow giant panda naturally revive.
The present invention is not limited to above embodiment, and LH peak value of the present invention and time of occurrence thereof are not necessarily found out by change curve intuitively, can only by more all detected LH value sizes, obtain.
Claims (2)
1. a determination methods of utilizing the in good time the time of artificial insemination of lutropin (LH) result optimizing giant panda, comprises the following steps:
(1) during breeding spring, by time requirement, Female Giant Panda is carried out to A urine sample collection device, then to urine sample, adopt elisa to detect Female Giant Panda estradiol real peak and time of occurrence thereof;
(2) take moment of occurring Female Giant Panda estradiol real peak as start time 0 hour, adopt elisa to detect in 0 hour to 48 hours all LH values of urine samples, find out LH peak value and time of occurrence thereof;
(3) according to LH peak value constantly or/and estradiol real peak constantly, LH peak value determines first the time of artificial insemination jointly with the peak behavior that whether occurs oestrusing of estradiol real peak interval, Female Giant Panda constantly constantly;
When Female Giant Panda LH peak value is constantly less than 16 hours with estradiol real peak interval constantly and when estradiol real peak constantly occurs oestrusing peak behavior with interior Female Giant Panda in latter 30 hours, can determine that Female Giant Panda is that normal ovulation is individual, therefore at LH peak value, in 14～30 hours after the moment and when Female Giant Panda occurs oestrusing peak behavior, be defined as first the time of artificial insemination;
When Female Giant Panda LH peak value constantly with estradiol real peak constantly interval greater than 16 hours and when estradiol real peak does not constantly occur oestrusing peak behavior with interior female individuals in latter 30 hours, can determine that Female Giant Panda is that delayed ovulation is individual, therefore in estradiol real peak, in 30～36 hours after the moment and when Female Giant Panda occurs oestrusing peak behavior, be defined as first the time of artificial insemination.
2. a kind of determination methods of utilizing the in good time the time of artificial insemination of lutropin (LH) result optimizing giant panda according to claim 1, is characterized in that: after first the time of artificial insemination, within 6～8 hours, be defined as the time of artificial insemination for the second time.
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