CN102743239B - Timely artificial insemination method for giant panda - Google Patents
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Abstract
The invention discloses a timely artificial insemination method for a giant panda. The method comprises the steps of: collecting, testing and analyzing urine samples of female giant pandas during oestrus in spring, simultaneously combining with these circumstances such as time that the drop of an estradiol maximal peak value or an estradiol peak value reaches up to over 50%, the condition whether a progesterone value is on the rise and the giant panda generates a typical heat peak behavior in the process of the estradiol dropping from the maximal peak value or subsequent peak value, and the ratio of the progesterone value corresponding to the estradiol maximal peak value or subsequent estradiol peak value to the progesterone basic value, judging a real peak value of the estradiol of the giant panda; then carrying out artificial insemination for the first time within 24-48 hours after the moment when the estradiol real peak value appears, and carrying out artificial insemination for the second time after 6-8 hours after finishing the artificial insemination for the second time, finally obviously improving the pregnancy rate of the giant panda. Compared with the past, the artificial insemination pregnancy rate of the giant panda is improved to 42.11% from 20.41% and is more than doubled.
Description
Technical field
The present invention relates to a kind of captive animal be carried out to test-tube technology, particularly relate to the in good time artificial insemination method of a kind of giant panda.
Background technology
Giant panda is the distinctive Rear Endangered Wildlife of China, and himself fertility is on the low side, Female Giant Panda 1 year for once oestrus, and only have 1～3 day in the oestrus, can accept natural mating, and its ovulation period may only have one day; The ratio of Captive Giant Panda natural mating is simultaneously on the low side, only has at present the giant panda energy natural mating of 50% left and right, there is the giant panda of half left and right to complete breeding by artificial insemination, because giant panda artificial insemination need to be carried out under narcotism, from available data and data, before ovulation, anesthesia can cause ovulation inhibition and the delayed ovulation of animal, if implement too early artificial insemination under narcotism before animal ovulation, can directly cause the decline of farrowing rate; On the other hand, the time that maintains insemination ability due to ovum after animal ovulation will be well below sperm, if spend evening and implement artificial insemination ovulation is rear, thereby ovum may be lost the decline that best fertilization causes farrowing rate opportunity because of aging.Therefore, giant panda the time of artificial insemination should be chosen in the rear reasonable time of ovulation and carry out just guaranteeing pregnancy rate theoretically, therefore determines that the in good time artificial insemination time of giant panda is the test-tube key technology of giant panda.
Before the mid-90 in last century, the judgement that giant panda is bred opportunity in good time mainly according to giant panda oestrus determination of the nature of the act observe, determine that by rule of thumb giant panda breeds opportunity in good time, cause the in good time artificial fertilization of the giant panda time to hold unclear, lack quantizating index, this is also to cause the low major reason of giant panda artificial insemination reproductive efficiency.
The journal > > of < < Sichuan University (natural science edition) 34:43～47 periodical in 1997 stepped on author for Feng Wen and, the different breeding wonies of the people's such as Zhang Anju article < < affect > > to giant panda farrowing effect, breeding result to 155 different reproductive modes of 1980～1993 years Captive Giant Panda is added up, find only 36% left and right of average pregnancy rate, and complete artificial insemination individuality be no matter to adopt fresh semen, frozen semen or both mix and use its pregnancy rate all lower, overall pregnancy rate only 20.4%, the results are shown in Table 1:
The comparison of the different breeding wony pregnancy rate of table 1.1980～1993 year Captive Giant Panda result
After the mid-90 in last century, by monitoring giant panda urine in oestrus estrogen Changing Pattern, the index of utilizing estrogen index in good time to breed as giant panda starts to be applied in giant panda artificial insemination gradually, but to adopting estrogen index to have very large dispute as the index of giant panda breeding in good time, there is no unified viewpoint.
Generally use at present elisa to detect giant panda urine sample estrogen.If using estrogen level peak value in giant panda urine, different research worker proposed different in good time breeding estrogen indexs as 0 day the same day.
(the Hodges JK such as Hodges, Bevan DJ, Celma M, Hearn JP, Jones DM, Kleiman DG, Knight JA, Moore DM.1984.Aspects of the reproductive endocrinology of the female Giant Panda (Ailuropoda melanoleuca) in captivity with special reference to the detection of ovulation and pregnancy.J.Zool., Lond203:253～267.) according to the result of study of 3 giant pandas, proposing in good time breeding time may be at the 1st day, the 2nd day.
Li Fudong, Peng Shiyuan, Ye Zhiyong, He Guangxin, the bright .1993 of Huang Xiang. in Female Giant Panda oestrus urine 17 beta estradiols and the variation of progesterone level and with the relation of breeding. beasts journal 13(3): 166-171, proposing in good time breeding time should be at the 0th day and the 1st day.
Li Xuebing, Huang Xiangming, Li Guanghan, Li Shaochang, Zhang Fuxiang, Yu Jianqiu, Zhong Shunlong, Song Yunfang, Chen Hongwei, Tang Fei, Nan Xi. the ELISA that Sha blocks Na .1994a. giant panda urine hormone in oestrus measures and in good time breeding. Chengdu international giant panda scientific seminar collection of thesis. Chengdu: the .247-250 of Sichuan science tech publishing house, think in good time breeding time should since breeding in 0 day always composite to lutropin LH peak value in urine, occur till.
The in good time the time of artificial insemination of giant panda that research worker proposes above, be all that to urinate estrogen peak value time of occurrence be reference, the in good time the time of artificial insemination of giant panda does not all have the test-tube example of enough successes to support that the correctness of its viewpoint is maximum problem.
From the success of having reported at present, carry out giant panda artificial insemination example, on the one hand the in good time the time of artificial insemination of giant panda is also lacked to more consistent view, and the test-tube individual amount of open report is less, thereby is difficult to fully support its viewpoint.
Yellow scorching .2001. use cryopreserved semen to carry out giant panda (Ailuropoda melanoleuca) artificial insemination. application and environmental organism journal-2001.7 (6) .-558-562, reported a routine giant panda artificial insemination case, think that the estrogen of the test-tube Best Times of giant panda in jenny urine reaches 48～72 hours after peak-peak, and after same author negated before propose this view.
Yellow scorching .2006. the research of Female Giant Panda estrone in oestrus. Sichuan Agricultural University's academic dissertation; by 2004～2005 years were detected 26 giant panda urine estrone content of China's protection giant panda research center; and natural mating and artificial insemination time of origin and successful relation of farrowing have been analyzed; proposing Female Giant Panda copulation or test-tube Best Times is the same day that urine estrone content reaches peak value, within 0 day, carries out.
The .2003 such as appearances are stated in analogy. idiophase Female Giant Panda urinate in the research of Hormone change. journal of animal science and veterinary medicine-2003.34 (5) .-452-456), the in good time breeding time of giant panda has been proposed again to different views, to 39 giant panda urine in oestrus estrone and after analyzing with farrowing result on breeding opportunity, proposing, in urine estrone peak value decline 12h, carry out natural mating first, artificial insemination or both alternately breedings, pregnancy rate reaches 100%; In peak value decline 12-24h, carry out natural mating first, artificial insemination or both alternately breedings, pregnancy rate reaches 83.3%; After peak value decline 24h, breed first, pregnancy rate is 0.
Although the artificial insemination case of rear two pieces of report reports is more, and by natural mating and the time of artificial insemination and the successful relation of farrowing, in good time breeding time has been proposed, but the greatest problem of rear two pieces of report existence is that two authors are all by these two kinds different breeding wonies merging statistics of artificial insemination and natural mating, and there is no to add up separately the relation of simple the time of artificial insemination and success farrowing.Because natural mating does not need anesthesia, impact on ovulation is less, artificial insemination need to be carried out under narcotism, if the time of artificial insemination is improper, to ovulation be produced to larger impact, therefore these two kinds different breeding wonies of artificial insemination and natural mating are merged to the in good time the time of artificial insemination proposing after statistics and can not truly reflect reasonably the time of artificial insemination in good time, therefore the time of artificial insemination that these two authors propose still lacks enough foundations, and the report that the in good time the time of artificial insemination proposing also lacks subsequent applications and farrowing rate result proves its correctness.
About the in good time technology of artificial insemination of existing giant panda, the in good time artificial insemination time proposing is at present all using urine estrogen peak value time of occurrence as basis for estimation, and the in good time artificial insemination time indicator difference proposing is larger.If occur that using estrogen peak value the same day was as 0 day, the in good time artificial insemination time of proposition is respectively: the first viewpoint is the 1st day, the 2nd day (Hodges etc., 1984); The second viewpoint is the 0th day and the 1st day (Li Fudong etc., 1993); The third viewpoint be since 0 day breeding always composite to urine in LH peak value occur till (Li Xuebing etc., 1994); The 4th kind of viewpoint is 0 day (Huang Yan, 2006); If the 5th kind of viewpoint is to occur that using estrogen peak value the moment was as 0 hour, breeding time is 0～24 hour in good time, is preferably 0～12 hour (appearance is stated in analogy, 2003).Summary is got up, if worthwhile day as 0 day using e surge, for the current viewpoint of artificial insemination time first, is 0 day or first day.
In sum, there is at present following problem in the judgment technology of the in good time the time of artificial insemination of existing giant panda: 1. current proposed all viewpoints all do not have practical application and obtain enough artificial insemination pregnancy rate (or farrowing rate) data to prove the correctness of its viewpoint.2. the suitable the time of artificial insemination that proposed only proposes based on several examples, lacks enough Data supports and causes being based on insufficient grounds; Or the in good time artificial insemination time proposing is not separated artificial insemination and natural mating, but natural mating and artificial insemination are merged to statistics proposition, there is no to add up separately simple test-tube opportunity and successful relation of farrowing, the time of artificial insemination that therefore proposed still lacks enough foundations.3. current proposed in good time artificial insemination viewpoint all proposes based on estrogen peak value time of occurrence, because giant panda heat Hormone change curve is originally as undulatory property curve, there will be multiple peak values, and giant panda ovulation also relates to progestogen, other the multiple factors such as behavior of oestrusing, iff can cause being difficult to judge the time of occurrence of real peak in practice according to estrogen result, but at present for how definitely judging whether estrogen peak value is that real peak there is no any relevant report, this is for determining in good time the time of artificial insemination and improving artificial insemination success rate and brought very big difficulty.
Summary of the invention
The technical problem to be solved in the present invention is a kind of how to judge giant panda estradiol real peak, the in good time artificial insemination method of giant panda how to carry out high-efficiency artificial insemination after giant panda estradiol real peak occurs.
For solving the problems of the technologies described above, the in good time artificial insemination method of a kind of giant panda of the present invention, comprises the following steps:
(1) spring during oestrus, by time requirement, Female Giant Panda is carried out to A urine sample collection device, observe the giant panda behavior of oestrusing, in urine sample, the freezing or stored refrigerated of the laggard row of labelling, then adopts elisa to detect estradiol value and progesterone value to urine sample simultaneously; The described labelling being recorded in urine sample mainly comprises that Female Giant Panda exhales name, urination time etc.; Described estradiol value and progesterone value refer to the estradiol value and the Concentration of Progesterone value result that by creatine concentration correcting determination in urine sample, obtain; Described estradiol is a kind of estrogen main in urine sample, and described progesterone is a kind of progestogen main in urine sample;
(2) using giant panda urination time as abscissa, using estradiol value and progesterone value on the plane coordinates of vertical coordinate, mark respectively urination time and corresponding estradiol value and the determined two groups of coordinate points of progesterone value thereof, two groups of coordinate points are connected in turn respectively, draw out estradiol value change curve and progesterone value change curve, find out estradiol peak-peak and corresponding progesterone value thereof; Described estradiol peak-peak is also the peak of estradiol;
(3) according to the progesterone value before estradiol peak-peak time of occurrence, calculate the Female Giant Panda progesterone basic value of oestrusing, and calculate estradiol peak-peak or progesterone value corresponding to estradiol peak value thereafter and the ratio of progesterone basic value;
(4) according to estradiol value change curve and progesterone value change curve, estradiol peak-peak or estradiol peak value are thereafter reached to the whether in rising trend and giant panda of time more than 50% used, estradiol progesterone value from the process that peak-peak or peak value are thereafter declining in fall and whether occur that typical case's situations such as the corresponding progesterone value of peak behavior, estradiol peak-peak or estradiol peak value thereafter and the ratio of progesterone basic value of oestrusing combine, and judge giant panda estradiol real peak jointly simultaneously; The described moment that occurs giant panda estradiol real peak is corresponding giant panda urination time;
(5) take estradiol real peak go out now as start time 0 hour, after the scope of 24 hours～48 hours in giant panda carry out artificial insemination for the first time while there is oestrusing peak behavior.
The described in good time artificial insemination method of a kind of giant panda, the meter of its Female Giant Panda progesterone basic value of oestrusing
Calculate and carry out according to the following steps:
1. choose all progesterone values before giant panda estradiol peak-peak, find out the maximum A in selected progesterone value, calculate arithmetic mean value B, the standard deviation C of selected progesterone value, then draw B+2C=D;
If 2. A-D is for negative, arithmetic mean value B is now progesterone basic value;
If 3. A-D, for just, finds out all progesterone values that are greater than D from the progesterone value of choosing, by the mode of 1. step, from residue progesterone value, find out maximum A
1, calculate the arithmetic mean value B that remains progesterone value
1, standard deviation C
1, then draw B
1+ 2C
1=D
1;
If 4. A
1-D
1still for just, repeat the 1. mode of step, until A
n-D
nfor negative, arithmetic mean value B now
nbe progesterone basic value, wherein n represents natural number.
The described in good time artificial insemination method of a kind of giant panda, if estradiol peak-peak fall within the scope of 10 hours～20 hours reaches more than 50%, estradiol drops to progesterone value corresponding to basic value, estradiol peak-peak and the ratio of progesterone basic value is greater than 2 continuously afterwards, the process simultaneously declining from peak-peak at estradiol, progesterone value corresponding to estradiol peak-peak is in rising trend there is typical case's peak behavior of oestrusing with giant panda, can determine that this estradiol peak-peak is panda estradiol real peak.
The described in good time artificial insemination method of a kind of giant panda, if estradiol peak-peak fall within the scope of 20 hours～31 hours reaches more than 50%, estradiol drops to progesterone value corresponding to basic value, estradiol peak-peak and the ratio of progesterone basic value is greater than 1.8 continuously afterwards, the process simultaneously declining from peak-peak at estradiol, progesterone value is in rising trend there is typical case's peak behavior of oestrusing with giant panda, can determine that this estradiol peak-peak is giant panda estradiol real peak.
The described in good time artificial insemination method of a kind of giant panda, if estradiol from peak-peak decline time of 50% more than 20 hours, progesterone value corresponding to estradiol peak-peak and the ratio of progesterone basic value is less than 1.8, after estradiol peak-peak, occur process that one or more peak values and estradiol decline from peak-peak that typical case's peak behavior of oestrusing does not appear in the not in rising trend and giant panda of progesterone value, can determine that estradiol peak-peak is false peaks simultaneously; Now, giant panda estradiol real peak is a certain peak value after estradiol peak-peak, and this peak value must possess simultaneously, and within the scope of 10 hours～20 hours fall reaches more than 50%, estradiol drops to that basic value, progesterone value that this peak value is corresponding are greater than 2 with the ratio of progesterone basic value, typical case's situations such as peak behavior of oestrusing appear in rising trend and giant panda in the estradiol progesterone value that this peak value is corresponding from the process of this peak value decline continuously afterwards.
10 hours～20 hours described scopes refer to that giant panda estradiol is from the decline main criterion of 50% time used of real peak, it is to sum up in the statistics by 78 routine giant panda heat glycol Changing Patterns, the data volume of 78 examples is considerably beyond any report amount in the past, it is maximum data volume in the world up to now, in 78 examples, there is 80% giant panda individuality all within the scope of this, the criterion of therefore, 10 hours～20 hours scopes has extreme high reliability.
The described giant panda typical case peak behavior of oestrusing comprises that (the special behavior of oestrusing appears to the reflection of obvious pressure tail in peak period giant panda oestrusing, with hard things such as bamboo poles, stimulating giant panda rear quarters, there is the quiet end reaction that stands, sticks up in giant panda) and behavior to be joined (quiet stand, stick up tail, trunk sinkage independently appear in giant panda).
The described in good time artificial insemination method of any giant panda, its process of Female Giant Panda being carried out to A urine sample collection device by time requirement is as follows:
(1) observe Female Giant Panda external genital, if external genital occur after redness, collect 1 part of Female Giant Panda urina sanguinis every day;
(2) the 4th day from estradiol rises continuously starts, and every day, 3 parts of urine samples were collected at interval about 8 hours;
(3) before and after Female Giant Panda is oestrused peak behavior, every day, 5 parts of urine samples were collected at interval about 5 hours;
(4) once monitor estradiol value, there is downward trend, A urine sample collection device is shortened to about 3 hours interval time.
The described in good time artificial insemination method of any giant panda, its each time giant panda A urine sample collection device comprise the following steps:
(1) clean and dry animal house hardened ground;
(2) giant panda of need being collected to urine sample is put into animal house, and staff waits for, once giant panda is discharged urine, immediately giant panda is put into other animal house;
(3) by clean injectors, gather the fresh urine sample in ground, pack cryovial into, carry out labelling, then put immediately freezing or cold storage plant in refrigerator.
The described in good time artificial insemination method of any giant panda, 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 cervical orifice;
(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.
The described in good time artificial insemination method of any giant panda, completes 6～8 in artificial insemination for the first time and as a child carried out afterwards artificial insemination for the second time.
Available data is without any report and correlation technique and criterion about how judging giant panda estrogen real peak, the present invention will solve the real peak that how to judge giant panda estradiol, determine criterion simultaneously, and giant panda urination time corresponding estradiol real peak is defined as to the time of occurrence of estradiol real peak.
Whether giant panda urine sample gathering can directly have influence on it interval time is the judgement of real peak, interval time, real peak value was missed in long meeting, interval time, too short one side can increase workload and the interference to animal, can reduce on the other hand the feasibility of sampling.Uncertain and disunity of giant panda urine sample gathering time of available data report, some acquisition times interval reaches 1 day, can cause the error in judgement scope of time to peak larger; And the sampling time interval scheme that the present invention adopts is about 3 hours in the peak period interval of oestrusing, make the error in judgement scope of giant panda estradiol time to peak narrow down to about 1.5 hours, greatly dwindled the error in judgement to giant panda estradiol time to peak.
Existing open report all detects merely urine estrogen level to the judgement time of giant panda estrogen peak value, and do not detect oestrus peak period progesterone level and behavioristics's index, comprehensively judges.Judge whether estradiol occurs the key of real peak is to judge whether estradiol occurs real decline, once estradiol appears in monitoring, decline, actually or still can not judge that estradiol fluctuation declines really.In order to address this problem, one aspect of the present invention is monitored urine progesterone level in monitoring urine estradiol, determines simultaneously and quantizes the true standard declining of estradiol, adopts behavioristics's supplementary means comprehensively to judge whether giant panda urine estradiol is true decline.
Judge whether estradiol occurs that the key of real peak is to judge whether estradiol occurs real decline, the present invention will be by statistical analysis giant panda heat as much as possible glycol data, determine that the decrease speed reference range of giant panda estradiol peak value quantizes to determine whether giant panda estradiol occurs true decline.
Once monitor giant panda, occur suspicious peak value, by strengthening sampling density by continuous sampling about sampling interval time shorten to 3 hour, the speed that monitoring estradiol declines determines whether estradiol is true decline.
Chengdu Panda Breeding Research Base has carried out statistical analysis to amounting to 78 routine giant panda estradiol Changing Patterns for many years, analyze estradiol from real peak more than 50% required time distribution that declines, draw out as shown in Figure 1 oestrus female giant panda estradiol from the real peak 50% required time distribution frequency that declines, adopt method of percentiles to determine the giant panda urine estradiol peak value 50% required time reference range that declines, the results are shown in Table 2:
Table 2. estradiol real peak 50% required time (h) that declines
As can be seen from Table 2 by the statistics of 78 routine giant panda heat glycol Changing Patterns is found, giant panda urine estradiol is that 31.17 hours, minima are 5.83 hours from the decline maximum of 50% required time of real peak, wherein 80% giant panda individuality is all within the scope of 9.83～19.86 hours, within the scope of 10～20 hours, be less than 20 hours; Analyze and occurred 16 routine giant pandas of estradiol false peaks simultaneously, analyze decline 50% required time and time thereof of estradiol peak value to distribute, the results are shown in Table 3:
Table 3. estradiol false peaks 50% required time (h) that declines
As can be seen from Table 3, for the individuality that occurs estradiol false peaks, estradiol will be longer than real peak value individuality from false peaks 50% required time that declines, within the scope of 14.58～41.5 hours, wherein 80% individuality, within the scope of 20～38.7 hours,, within the scope of 20～39 hours, is greater than 20 hours.
Utilize these data to judge that to differentiating giant panda urine estradiol is that fluctuation or true decline provide strong quantification reference data, once monitoring giant panda urine estradiol occurs declining rapidly from a high value at short notice, if estradiol fall reaches more than 50% in the scope of 10 hours～20 hours, can judge that this reduces to true decline; Once and occur that estradiol decrease speed is excessively slow, time of 50% of decline exceedes 20 hours, can tentatively be judged as estradiol fluctuation, but not truly declines.
Existing, about giant panda, in good time test-tube open report, only adopt giant panda estrogen peak value as a reference, do not monitored urine sample progesterone level simultaneously.The present invention is when detecting giant panda urine sample estradiol, monitor giant panda urine sample progesterone level simultaneously, once there is the decline of estradiol, check that progesterone level changes simultaneously, if progesterone is without ascendant trend, be judged as the fluctuation of estradiol, but not truly decline, if progesterone occur ascendant trend estradiol may there is true decline, by the acquisition interval time that further shortens urine sample to every about 3 hours once, until the true fall time of full confirmation estradiol, thereby improved the accuracy that estradiol detects.
In order to quantize to determine giant panda corresponding progesterone level difference while there is true and false estradiol peak value, in order to further quantification, determine estradiol real peak, compared the difference of the ratio of progesterone value that true and false estradiol peak value is corresponding and progesterone basic value, the results are shown in Table 4:
The progesterone value that the true and false estradiol peak value of table 4. is corresponding and the odds ratio of progesterone basic value are
As can be seen from Table 4, in 80% individuality when there is estradiol real peak, and its corresponding progesterone value level raises, and the ratio of progesterone value and its basic value, between 1.79～3.14, is greater than 1.8; And when occurring that, after false estradiol peak value, its corresponding progesterone value level is lower, 80% individual progesterone value and the ratio of its basic value, between 0.94～2.13, are less than 2, these data can further quantize to determine whether estradiol is real peak.If occur after suspicious estradiol peak value, calculate the ratio of its corresponding progesterone value and progesterone basic value, if be greater than 2, can further confirm that this estradiol peak value is real peak, if this ratio is less than 1.8, this estradiol peak value may be false peaks, if be greater than 1.8, need to determine further combined with behavioristics's index.
In existing open report, less than oestrusing about employing giant panda, whether behavior auxiliary judgment estradiol is the true relevant report declining, only Science Press in 2006, publish, the page number is 257 pages, author for Zhang Zhi and with the < < giant panda in situ conservation theory and practice > > of Wei Fuwen in have related description: the time of origin of 86.4% (19/22) giant panda natural mating is first after estrogen peak value, the time that giant panda can carry out natural mating appears at the peak of oestrusing conventionally.This < < giant panda in situ conservation theory and practice > > shows, can assist and adopt giant panda whether to occur oestrusing peak behavior as the whether truly auxiliary reference foundation of decline of estrogen.
Once monitoring giant panda, the present invention occurs that estradiol declines, progesterone rises, observe the giant panda behavior of oestrusing simultaneously, if there is typical case's peak behavior of oestrusing in giant panda, can occur that estradiol truly declines by auxiliary judgment giant panda, otherwise still can not full confirmation, this will further get rid of estradiol and progesterone and occur simultaneously the situation of fluctuation.
Because giant panda heat glycol presents fluctuation-type change curve, once and there is estradiol peak value, its decrease speed is very fast, due to impossible continuous acquisition urine sample on giant panda, there is certain time interval in sampling, although strengthened sample frequency, still may miss real estradiol peak value sample,, there is false peaks in the but situation of real estradiol peak value not necessarily of the estradiol peak-peak that causes part individuality there will be to see from numerical value.In existing all open reports, all less than the technology contents about how rejecting false peaks.
Existing technology only single dependence estrogen index judges the time of artificial insemination, has very large limitation, is difficult to definitely hold the time of artificial insemination; The present invention will adopt estradiol, progesterone, three index comprehensives of behavior of oestrusing to judge suitable artificial fertilization's opportunity.
Estradiol index: determine estradiol real peak time of occurrence according to the present invention, using the estradiol real peak moment as 0 hour, the in good time breeding time of giant panda is between 24～48 hours.
Progesterone index: if using the estradiol real peak moment as 0 hour, the in good time breeding time of giant panda need carry out between 24～48 hours and when progesterone is in rising trend, if do not occur, progesterone rises, and is not suitable for doing artificial insemination.
Behavioristics's index: if using the estradiol real peak moment as 0 hour, the suitable artificial insemination time first of giant panda is between 24～48 hours, and need to be chosen in when giant panda occurs oestrusing typical case peak behavior and progesterone rise and carry out.
Owing to not generally acknowledging at present the definite in good time artificial insemination time of giant panda, therefore in giant panda artificial insemination practice, artificial insemination time changes first, if occur that using estrogen peak value the same day was as 0 day, even using estrogen peak value, occur that the moment was as 0 hour, at present the giant panda of open report first artificial insemination time greatly within the scope of 0～48 hour.
In order to determine in good time artificial insemination time, Chengdu Panda Breeding Research Base was from 2005～2010 years random selected part health, of the right age (6～19 years old) individualities giant panda artificial insemination, carry out early stage artificial insemination (using the estradiol real peak moment as 0 hour, first artificial insemination time was carried out in 0～24 hour), and by early stage artificial insemination group as a control group; Simultaneously random selected part health, of the right age giant panda carry out artificial insemination in late period (using the estradiol real peak moment as 0 hour, artificial insemination time is in 24～48 hours first, occur when progesterone rises and occurs oestrusing peak behavior carrying out), the farrowing of statistics giant panda, miscarriage situation, to farrow and miscarry and be individual individual as gestation, otherwise as gestation is not individual, calculate pregnancy rate (pregnant tire number/artificial insemination number of individuals * 100%), two groups of giant panda pregnancy rate are compared, the results are shown in Table 5:
The comparison of the different the time of artificial insemination giant panda of table 5. pregnancy rate
| Grouping | Gestation number of individuals | Not pregnant number of individuals | Artificial insemination number of individuals | Pregnancy rate % |
| Early stage artificial insemination group | 7 | 10 | 17 | 41.18 |
| Artificial insemination in late period group | 7 | 7 | 14 | 50.00 |
| Add up to | 14 | 17 | 31 | 45.16 |
As can be seen from Table 5, by upper table, can see, no matter early stage or carry out artificial insemination late period, all can successful reproduction offspring, but late period test-tube pregnancy rate higher than in early days.Between 24～48 hours after the estradiol real peak moment, when progesterone rises and occur oestrusing peak behavior, carry out first artificial insemination, its pregnancy rate was higher than 0～24 hour.To the of the right age healthy giant panda of 14 example, adopt technology of artificial insemination to carry out artificial insemination, farrowing rate reaches 50%, and artificial insemination in late period group reproductive efficiency further improves, and the more early stage artificial insemination group of pregnancy rate has improved 8.82%.By the above results, can draw: if using the estradiol real peak moment as 0 hour, the suitable artificial insemination time first of giant panda, in 24～48 hours, is carried out when progesterone rises and occur oestrusing peak behavior.
This viewpoint has following features compared with existing open source information:
First, the in good time the time of artificial insemination proposing in all open reports is at present only based on 1～3 artificial insemination example or natural mating and artificial insemination are not separated to statistics, and the time of artificial insemination of proposition lacks enough foundations; And the time of artificial insemination that the present invention proposes gathers more artificial insemination example, natural mating and artificial insemination case are separated simultaneously, and only adopt of the right age (6～19 years old), the individual complete artificial insemination analysis of cases of Healthy female, got rid of the improper reproductive failure causing of through poor health and age, and in good time the time of artificial insemination is proposed, the time of artificial insemination that therefore proposed is according to more abundant.
Secondly, if using the estradiol real peak moment as 0 hour, it is 24～48 hours that the present invention proposes the in good time artificial insemination time of giant panda first, all different from existing open source information viewpoint, existing open source information all not by giant panda first artificial insemination time be defined as after 24 hours, so in fact, negated the past in good time giant panda artificial insemination time to be defined as 0 day or the viewpoint of first day, in fact postponed the first artificial insemination time of giant panda.
Meanwhile, the present invention, when determining suitable first the time of artificial insemination, is chosen in while there is oestrusing peak behavior between the progesterone rising stage simultaneously and carries out, for the present invention proposes and Successful Practice first.
Finally, the in good time artificial insemination time that the present invention proposes with hour rather than take sky as unit, more accurate and be easy to hold, the in good time artificial insemination times of the most propositions of existing open source information are unit take sky, time ambiguity, is difficult to definite assurance.
In openly reporting about giant panda artificial insemination after 2000, maximum 7 routine giant panda artificial insemination case (Huang Yan that only reported, 2002), be difficult to illustrate test-tube pregnancy rate, only have at present Feng Wenhe (Feng Wen and, Zhang Anju, 1997) 1980～1993 years were amounted to 98 time giant panda artificial insemination pregnancy rate are added up.Because the enforcement artificial fertilization's of Feng Wen and statistics individuality is not by health problem and non-of the right age individual eliminating, therefore, for compare with past data as far as possible, since 2005～2010 years, in Chengdu Panda Breeding Research Base, to including health problem, age 19 giant pandas between 5 years old～22 years old, implemented the present invention, the data of experimental result and Feng Wen and report contrast, and comparing result is in Table 6:
The comparison of the different technology of artificial insemination farrowing rate of table 6.
| Grouping | Gestation | Not gestation | Artificial insemination number of individuals | Pregnancy rate |
| Feng Wen and report | 20 | 78 | 98 | 20.41% |
| The technology of the present invention | 8 | 11 | 19 | 42.11%* |
| Add up to | 28 | 89 | 117 | 23.93% |
Remarks: * represents significant difference (p=0.042<0.05)
Adopt SPSS statistical software with X 2 test, to carry out diversity ratio to the giant panda artificial insemination pregnancy rate data acquisition of pregnancy rate of the present invention and Feng Wen and report, probability of outcome P=0.042<0.05, probability P <0.05 represents significant difference, and the result comparison that adopts the technology of the present invention to implement artificial insemination and Feng Wen and report can significantly improve giant panda pregnancy rate.
Because the present invention can accurately establish giant panda estradiol real peak and gone out now, and then go out now as start time 0 hour take giant panda estradiol real peak, after 24～48 hours in occur that progesterone rises and carries out artificial insemination for the first time while oestrusing peak behavior, after completing 6～8 hours, artificial insemination for the first time carries out again artificial insemination for the second time, final giant panda pregnancy rate has obtained and has significantly improved, and giant panda artificial insemination pregnancy rate is by 20.41% bringing up to 42.11%, having turned over more than one times compared with in the past.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 be oestrus of the present invention female giant panda estradiol from the real peak 50% time distribution frequency schematic diagram used that declines;
Fig. 2 is the change curve of the first estradiol value and the progesterone value of Female Giant Panda individuality of the present invention;
Fig. 3 is the change curve of the second estradiol value and the progesterone value of Female Giant Panda individuality of the present invention;
Fig. 4 is the third estradiol value of Female Giant Panda individuality of the present invention and the change curve of progesterone value.
In figure, identify: α is the coordinate points of giant panda estradiol real peak on estradiol value change curve, β is the coordinate points corresponding with α on progesterone value change curve, Ec ng/mg Cr is expressed as the estradiol concentration value result obtaining by creatine concentration correcting determination in urine sample, and Pg ng/mg Cr is expressed as the Concentration of Progesterone value result obtaining by creatine concentration correcting determination in urine sample.
The specific embodiment
The present invention spring giant panda during oestrus observe Female Giant Panda pudendum color every day, the feature performance that giant panda pudendum redness starts for giant panda oestruses, once find pudendum redness, can tentatively determine that giant panda enters oestrus, continue to observe the giant panda behavior of oestrusing every day, if occur, frequent marking behavior, baa can determine that behavior giant panda oestruses.
After giant panda oestruses and starts, start Female Giant Panda urine sample to collect, in urine sample, recording panda exhales name, urination time, whether occurs typical case's labellings such as peak behavior of oestrusing, and in time the giant panda urine sample of collecting is carried out to assay, adopt elisa to detect with giant panda and exhale name and corresponding estradiol value and the progesterone value of urination time; Originally collect 1 part of urina sanguinis every day; After estradiol value rises continuously, within the 4th day, start, collect 3 parts of urine samples every day, interval is about 8 hours, and the time is respectively 7:00,15:00,23:00 left and right; Oestrus before and after peak, every day, urine sample was increased to 5 left and right, and interval is about 5 hours, and acquisition time is respectively: 6:00,11:00,16:00,21:00,1:00 left and right; Once monitor estradiol value, there is downward trend, A urine sample collection device is shortened to about 3 hours interval time, until finally confirm the true decline of estradiol value.
Giant panda A urine sample collection device is all the hardened ground collection from clean each time, first cleans and dries behind animal house ground, need is collected to the giant panda of urine sample and is put into animal house; Then staff waits for, once giant panda is discharged urine, immediately giant panda is put into other animal house, by the fresh urine sample in clean injectors collection ground, packs cryovial into, carries out labelling, puts into immediately the freezing preservation of freezer compartment of refrigerator or cold room stored refrigerated.
Using giant panda urination time as abscissa, using estradiol value and progesterone value on the plane coordinates of vertical coordinate, mark respectively urination time and corresponding estradiol value and the determined two groups of coordinate points of progesterone value thereof, two groups of coordinate points are connected in turn respectively, draw out estradiol value change curve and progesterone value change curve, find out estradiol peak-peak or progesterone value corresponding to estradiol peak value thereafter, calculate the Female Giant Panda progesterone basic value of oestrusing.
The calculating of the Female Giant Panda progesterone basic value of oestrusing is carried out according to the following steps:
1. choose progesterone value corresponding to all giant panda estradiol values before giant panda estradiol peak-peak, find out the maximum A in selected progesterone value, calculate arithmetic mean value B, the standard deviation C of selected progesterone value, then draw B+2C=D;
If 2. A-D is for negative, arithmetic mean value B is now progesterone basic value;
If 3. A-D, for just, finds out all progesterone values that are greater than D from the progesterone value of choosing, by the mode of 1. step, from residue progesterone value, find out maximum A
1, calculate the arithmetic mean value B that remains progesterone value
1, standard deviation C
1, then draw B
1+ 2C
1=D
1;
If 4. A
1-D
1still for just, repeat the 1. mode of step, until A
n-D
nfor negative, arithmetic mean value B now
nbe progesterone basic value, wherein n is natural number.
According to estradiol value change curve and progesterone value change curve, estradiol peak-peak or estradiol peak value are thereafter reached to the whether in rising trend and giant panda of time more than 50% used, estradiol progesterone value from the process that peak-peak or peak value are thereafter declining in fall and whether occur that typical case's situations such as the corresponding progesterone value of peak behavior, estradiol peak-peak or estradiol peak value thereafter and the ratio of progesterone basic value of oestrusing combine, and judge giant panda estradiol real peak jointly simultaneously.The method that judges giant panda estradiol real peak has respectively following three kinds of situations.
The first situation: as shown in Figure 2, if estradiol peak-peak fall within the scope of 10 hours～20 hours reaches more than 50%, estradiol drops to progesterone value corresponding to basic value, estradiol peak-peak and the ratio of progesterone basic value is greater than 2 continuously afterwards, the process simultaneously declining from peak-peak at estradiol, progesterone value is in rising trend there is typical case's peak behavior of oestrusing with giant panda, can determine that this estradiol peak-peak is giant panda estradiol real peak, be the α coordinate points in Fig. 2, β is the progesterone value coordinate points corresponding with α.
The second situation: as shown in Figure 3, if estradiol peak-peak fall within the scope of 20 hours～31 hours reaches more than 50%, estradiol drops to progesterone value corresponding to basic value, estradiol peak-peak and the ratio of progesterone basic value is greater than 1.8 continuously afterwards, the process simultaneously declining from peak-peak at estradiol, progesterone value is in rising trend there is typical case's peak behavior of oestrusing with giant panda, can determine that this estradiol peak-peak is giant panda estradiol real peak, be the α coordinate points in Fig. 3, β is the progesterone value coordinate points corresponding with α.
The third situation: as shown in Figure 4, if estradiol from peak-peak decline time of 50% more than 20 hours, progesterone value corresponding to estradiol peak-peak and the ratio of progesterone basic value is less than 1.8, after estradiol peak-peak, occur process that one or more peak values and estradiol decline from peak-peak that typical case's peak behavior of oestrusing does not appear in the not in rising trend and giant panda of progesterone value, can determine that this estradiol peak-peak is false peaks simultaneously; Now, giant panda estradiol real peak is a certain peak value after estradiol peak-peak, and this peak value must possess the in rising trend and giant panda of within the scope of 10 hours～20 hours fall reaches more than 50%, estradiol drops to that basic value, progesterone value that this peak value is corresponding are greater than 2 with the ratio of progesterone basic value continuously afterwards, this peak value is corresponding the process that declines from this peak value at estradiol progesterone value simultaneously and occur typical case's situations such as peak behavior of oestrusing, be the α coordinate points in Fig. 4, β is the progesterone value coordinate points corresponding with α.
The estradiol real peak that above three kinds of situations are judged respectively go out to be defined as now start time 0 hour, be that corresponding giant panda urination time is 0 hour, after the scope of 24 hours～48 hours in occur that progesterone rises and carries out artificial insemination for the first time while oestrusing peak behavior, artificial insemination for the first time completes carries out artificial insemination for the second time again after 6～8 hours.
Each artificial insemination all occurs that giant panda oestrus peak behavior and progesterone value of typical case carry out when in rising trend, and operation 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 urethral orifice and cervical orifice;
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 can utilize seme-deposition device before artificial insemination, inserts Female Giant Panda urethral orifice, draws and discharge the long-pending urine of intravesical.
Claims (6)
1. a determination methods for the in good time the time of artificial insemination of giant panda, comprises the following steps:
(1) spring during oestrus, by time requirement, Female Giant Panda is carried out to A urine sample collection device, observe the giant panda behavior of oestrusing, in urine sample, the freezing or stored refrigerated of the laggard row of labelling, then adopts elisa to detect estradiol value and progesterone value to urine sample simultaneously;
(2) using giant panda urination time as abscissa, using estradiol value and progesterone value on the plane coordinates of vertical coordinate, mark respectively urination time and corresponding estradiol value and the determined two groups of coordinate points of progesterone value thereof, two groups of coordinate points are connected in turn respectively, draw out estradiol value change curve and progesterone value change curve, find out estradiol peak-peak time of occurrence and corresponding progesterone value thereof;
(3) according to the progesterone value before estradiol peak-peak time of occurrence, calculate the Female Giant Panda progesterone basic value of oestrusing, and calculate estradiol peak-peak or progesterone value corresponding to estradiol peak value thereafter and the ratio of progesterone basic value;
(4) according to estradiol value change curve and progesterone value change curve, estradiol peak-peak or estradiol peak value are thereafter reached to the whether in rising trend and giant panda of time more than 50% used, estradiol progesterone value from the process that peak-peak or peak value are thereafter declining in fall and whether occur that the oestrus ratio of peak behavior, estradiol peak-peak or the corresponding progesterone value of estradiol peak value thereafter and progesterone basic value of typical case combines, and judges giant panda estradiol real peak jointly simultaneously;
(5) take estradiol real peak go out now as start time 0 hour, after the scope of 24 hours～48 hours in, occurring that progesterone is defined as the time of artificial insemination for the first time when rising and occurring oestrusing peak behavior.
2. the determination methods of the in good time the time of artificial insemination of a kind of giant panda according to claim 1, it is characterized in that: if estradiol peak-peak fall within the scope of 10 hours～20 hours reaches more than 50%, estradiol drops to progesterone value corresponding to basic value, estradiol peak-peak and the ratio of progesterone basic value is greater than 2 continuously afterwards, the process simultaneously declining from peak-peak at estradiol, progesterone value is in rising trend there is typical case's peak behavior of oestrusing with giant panda, can determine that this estradiol peak-peak is giant panda estradiol real peak.
3. the determination methods of the in good time the time of artificial insemination of a kind of giant panda according to claim 1, it is characterized in that: if estradiol peak-peak fall within the scope of 20 hours～31 hours reaches more than 50%, estradiol drops to progesterone value corresponding to basic value, estradiol peak-peak and the ratio of progesterone basic value is greater than 1.8 continuously afterwards, the process simultaneously declining from peak-peak at estradiol, progesterone value is in rising trend there is typical case's peak behavior of oestrusing with giant panda, can determine that this estradiol peak-peak is giant panda estradiol real peak.
4. the determination methods of the in good time the time of artificial insemination of a kind of giant panda according to claim 1, it is characterized in that: estradiol from peak-peak decline time of 50% more than 20 hours, progesterone value corresponding to estradiol peak-peak and the ratio of progesterone basic value is less than 1.8, after estradiol peak-peak, occur process that one or more peak values and estradiol decline from peak-peak that typical case's peak behavior of oestrusing does not appear in the not in rising trend and giant panda of progesterone value, can determine that this estradiol peak-peak is false peaks simultaneously; Now, giant panda estradiol real peak is a certain peak value after estradiol peak-peak, and this peak value must possess simultaneously, and within the scope of 10 hours～20 hours fall reaches more than 50%, estradiol drops to that basic value, progesterone value that this peak value is corresponding are greater than 2 with the ratio of progesterone basic value, typical case's peak behavior of oestrusing appears in rising trend and giant panda in the estradiol progesterone value that this peak value is corresponding from the process of this peak value decline continuously afterwards.
5. according to the determination methods of the in good time the time of artificial insemination of a kind of giant panda described in claim 1～4, its process of Female Giant Panda being carried out to A urine sample collection device by time requirement is as follows:
(1) observe Female Giant Panda external genital, if external genital occur after redness, collect 1 part of Female Giant Panda urina sanguinis every day;
(2) the 4th day from estradiol rises continuously starts, and every day, 3 parts of urine samples were collected at interval about 8 hours;
(3) before and after Female Giant Panda is oestrused peak behavior, every day, 5 parts of urine samples were collected at interval about 5 hours;
(4) once monitor estradiol value, there is downward trend, A urine sample collection device is shortened to about 3 hours interval time.
6. the determination methods of the in good time the time of artificial insemination of a kind of giant panda according to claim 5, its each time giant panda A urine sample collection device comprise the following steps:
(1) clean and dry animal house hardened ground;
(2) giant panda of need being collected to urine sample is put into animal house, and staff waits for, once giant panda is discharged urine, immediately giant panda is put into other animal house;
(3) by clean injectors, gather the fresh urine sample in ground, pack cryovial into, carry out labelling, then put into immediately freezing or cold storage plant.
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| CN103126783B (en) * | 2013-03-06 | 2014-05-07 | 成都大熊猫繁育研究基地 | Method for optimizing timely artificial insemination for pandas by aid of LH (luteinizing hormone) results |
| CN103207280B (en) * | 2013-03-21 | 2014-01-01 | 成都大熊猫繁育研究基地 | Method for predicting panda expected delivery date |
| CN115089339B (en) * | 2022-08-11 | 2023-05-16 | 成都大熊猫繁育研究基地 | Prediction method for estrus starting time of pandas |
| CN115561444B (en) * | 2022-11-30 | 2023-03-14 | 成都大熊猫繁育研究基地 | Method for early predicting peak occurrence time of estrone in pandas |
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