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1 FIXED-TIME INSEMINATION IN THE PIG 猪的定时输精 STUDIES AND APPLICATION WITH RESPECT TO ESTRUS SYNCHRONIZATION AND OVULATION INDUCTION 同期发情和诱导排卵的研究和应用 Prof. Dr. Dr. h.c. KLAUS-PETER BRÜ SSOW

2 The basic principles of artificial insemination (AI) are simply - i.e. to deposit enough viable sperm in the appropriate place and at the correct time relative to the time of ovulation. 人工授精的基本原理很简单, 即在相对排卵的合适时机和合适的位置输送足够多有活力的精子 However, the onset of estrus and the time of ovulation within a group of sows can varying widely. 但一组母猪常常发情开始时间和排卵时间不同 2

3 Therefore, directing estrus and ovulation of sows within a group would then allow for (group wise) fixed-time insemination. 故一批母猪发情和排卵, 可允许批次定时输精 This is of great benefit for swine farms that operate using sow batch management. It helps to savings in labor and to produce large batches of evenly developed pigs. 这对实施母猪生产批次化管理的猪场非常有利它有助于节省劳动力和生产大批量均衡生长的猪 3

4 If ovulation induction with fixed time AI should work optimally, several steps must occur: 定时输精的条件 (1) follicle growth and maturation should be synchronized in all females 所有母猪卵泡发育和成熟必须同步化 (2) all females would have mature follicles at time of induction 所有母猪诱导排卵时有成熟卵泡 (3) all would have a similar timed LH surge inducing ovulation 所有诱导排卵的母猪有定时 LH 峰 (4) all follicles would ovulate in a defined time window 在一个限定的时间内所有卵泡排卵 (5) sperm would be established in the oviductal reservoir before ovulation to fertilize all eggs. 排卵前精子确保在输卵管汇集 4

5 Some introducing notes to understand follicle development and hormonal regulation of estrus and ovulation in pigs 猪卵泡发育发情和排卵的激素调控原理 + - GnRH HYPOTHALAMUS 下丘脑 PITUITARY 垂体 + - FSH LH GONADS 性腺 + - ESTRADIOL / PROGESTERON 雌激素 / 孕激素 5

6 FOLLICLE DIAMETER (mm) 卵泡直径 (mm) FOLLICULAR GROWTH DURING ESTRUS CYCLE AND PREGNANCY 发情周期和妊娠期的卵泡发育 10 LUTEAL PHASE / SUCKLING PERIOD 黄体期 / 哺乳期 CORPORA LUTEA 黄体 FOLLICLE PHASE / AFTER WEANING 卵泡期 / 断奶后 6 4 DAYS OF EC 发情周期 DAYS AFTER WEANING 断奶后天数

7 HORMONAL CONTROL OF FOLLICLE MATURATION AND OVULATION 卵泡成熟和排卵的激素调控 + - GnRH HYPOTHALAMUS 下丘脑 + - FSH PITUITARY 垂体 LH GONADS 性腺 + - ESTRADIOL / PROGESTERON 雌激素 / 孕激素 6

8 Gonadotropin-Releasing Hormone (GnRH) 促性腺激素释放激素 Decapeptid; synthesized in the hypothalamus; causes synthesis and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary gland. 十肽, 下丘脑合成的, 促进垂体前叶合成和释放促黄体素 (LH) 和促卵泡素 (FSH) + - GnRH FSH HYPOTHALAMUS PITUITARY LH GONADS ESTRADIOL / PROGESTERON LH - LUTEINIZING HORMONE LH- 促黄体素 secreted from the pituitary; directs genesis of steroids and gametes; induces ovulation 垂体分泌, 直接促进类固醇激素合成和诱导排卵 FSH FOLLICLE-STIMULATING HORMONE FSH- 促卵泡素 promotes follicle recruitment and growth 促进卵泡募集和生长 ESTRADIOL-17ß (E2) 雌二醇 main ovarian steroid of the maturing follicle (but also secreted by the corpus luteum (CL) 主要由成熟卵泡分泌的类固醇 ( 黄体也能分泌 ) PROGESTERONE (P4) 孕酮 main ovarian steroid secreted by the CL (but also secreted by the pre-ovulatory follicle) 主要由黄体产生 ( 排卵前卵泡也能分泌 ) 8

9 OVULATION 排卵 OVULATION 排卵 OVARIAN DEVELOPMENT AND HORMONE SECRETION DURING ESTRUS CYCLE 发情周期中卵巢发育和激素分泌 OVARY FSH LUTEAL PHASE 黄体期 FOLLICLE PHASE 卵泡期 LH E 2 P 4 ESTRUS DAY OF ESTRUS CYCLE ESTRUS HEAT 9

10 OVULATION OVARIAN DEVELOPMENT AND HORMONE SECRETION AFTER WEANING 断奶后卵巢发育和激素分泌 SUCKLING / WEANING FOLLICLE GROWTH OVARY FSH LH E 2 P 4 DAY AFTER WEANING ESTRUS HEAT 11

HOW TO SYNCHRONIZE FOLLICLE DEVELOPMENT AND ESTRUS IN GROUPS OF GILTS AND SOWS WITH RESPECT TO FIXED-TIME AI 如何使定时输精的后备母猪和经产母猪卵泡发育同步和同期发情 SOWS : GROUP-WISE WEANING 批次断奶 GILTS :

11 HOW TO SYNCHRONIZE FOLLICLE DEVELOPMENT AND ESTRUS IN GROUPS OF GILTS AND SOWS WITH RESPECT TO FIXED-TIME AI 如何使定时输精的后备母猪和经产母猪卵泡发育同步和同期发情 SOWS : GROUP-WISE WEANING 批次断奶 GILTS : MANIPULATION / SYNCHRONIZATION OF ESTRUS CYCLE 同期发情 ESTRUS 发情期 PRO- & POST- ESTRUS 发情前期 DIESTRUS 发情间期 NON-INFLUENCED ESTROUS CYCLE SUCKLING PERIOD SYNCHRONIZATION SYNCHRONIZED ESTRUS 11

HOW TO ARRANGE SYNCHRONIZATION OF ESTRUS IN GILTS 如何使后备母猪同期发情 Suppression of follicular development by application of steroidal and non-steroidal substances: 通过使用来固醇或者非类固醇类物质抑制卵泡发育 Feeding of

12 HOW TO ARRANGE SYNCHRONIZATION OF ESTRUS IN GILTS 如何使后备母猪同期发情 Suppression of follicular development by application of steroidal and non-steroidal substances: 通过使用来固醇或者非类固醇类物质抑制卵泡发育 Feeding of methallibure 饲喂美他硫脲 (GERRITS & JOHNSON 1965, POLGE et al. 1968, BERGFELD et al. 1976) X Feeding of allyltrenbolone (altrenogest; REGUMATE, MATRIX ) 饲喂烯丙孕素 (KNIGHT et al. 1976, DAVIS 1976, MARTINAT-BOTTE 1990, HÜ HN et al. 1996) Application of progesterone or GnRH via slow-release formula 使用孕酮或 GnRH 缓释制剂 (PEACOCK et al. 1992, ESTILL et al. 1997, BRÜ SSOW et al. 2011)? 12

HOW TO ARRANGE SYNCHRONIZATION OF ESTRUS IN GILTS 如何使后备母猪同期发情 Influencing the Corpus luteum (CL) function 影响黄体功能 Extending CL lifespan by application of gonadotropin (hcg) 应用 hcg 延长黄体寿命 (GUTHRIE &

13 HOW TO ARRANGE SYNCHRONIZATION OF ESTRUS IN GILTS 如何使后备母猪同期发情 Influencing the Corpus luteum (CL) function 影响黄体功能 Extending CL lifespan by application of gonadotropin (hcg) 应用 hcg 延长黄体寿命 (GUTHRIE & REXROAD 1981, GUTHRIE & BOLDT 1983)? Prolonging the CL function by application of estrogens 应用雌激素延长黄体功能 (GEISERT et al.1987)? Induction and luteolysis of CL by reciprocal injection of gonadotropins and prostaglandins 促性腺激素和前列腺素配合用药诱导黄体溶解 (GUTHRIE & POLGE 1976, GUTHRIE 1979, SCHNURRBUSCH et al. 1993)? 13

follicles < 5 mm remains) 孕激素样活性, 抑制排卵卵泡生长 ( 卵泡大小 <5mm) imitation of luteal phase 模仿黄体期 RECOMMENDATION FOR APPLICATION 推荐使用方法

14 BIOLOGICAL ACTIVE SUBSTANCE TO SYNCHRONIZE ESTRUS IN GILTS 后备母猪同期发情的药品 REGUMATE, MATRIX active substance: altrenogest synthetic progestagen 活性成分 : 烯丙孕素 - 合成的孕激素 progesterone-like activity, suppresses growth of ovulatory competent follicles (growth of follicles < 5 mm remains) 孕激素样活性, 抑制排卵卵泡生长 ( 卵泡大小 <5mm) imitation of luteal phase 模仿黄体期 RECOMMENDATION FOR APPLICATION 推荐使用方法 FRANCE 法国 : GERMANY 德国 : NORTHERN AMERICA 北美 : 20 mg/day/gilt for 18 d 20 mg/day/gilt for 18 d or 16 mg/day/gilt for 15 d 15 mg/day/gilt for 14 d 14

15 FOLLICLE DIAMETER (mm) 卵泡直径 (mm) STIMULATION OF FOLLICLE GROWTH 刺激卵泡发育 GONADOTROPINS (GONA) STIMULATE GROWTH OF OVULATORY 促性腺激素促进排卵卵泡发育 10 REGUMATE FEEDING/ SUCKLING WEANING 饲喂烯丙孕素 / 哺乳 - 断奶 GONA FOLLICLE PHASE / AFTER WEANING 卵泡期 / 断奶后 6 4 DAYS OF EC DAYS AFTER WEANING

BIOLOGICAL ACTIVE SUBSTANCE TO STMULATE FOLLICLE GROWTH 促进卵泡发育的药物 ecg equine Chorion Gonadotropin 马绒毛膜促性腺激素 (PMSG pregnant mares serum gonadotropin 孕马血清促性腺激素 ) high purified serum preparation of

16 BIOLOGICAL ACTIVE SUBSTANCE TO STMULATE FOLLICLE GROWTH 促进卵泡发育的药物 ecg equine Chorion Gonadotropin 马绒毛膜促性腺激素 (PMSG pregnant mares serum gonadotropin 孕马血清促性腺激素 ) high purified serum preparation of pregnant mares 高纯度 gonadotropin with both FSH and LH activity (FSH/LH ratio about 0.2 : 1) 有 FSH 和 LH 两种活性 (FSH/LH 约为 0.2:1) stimulates follicular development and final follicle maturation 刺激卵泡发育和卵泡最终成熟 stimulates onset of estrus, pre-ovulatory LH release and ovulation 刺激发情, 排卵前 LH 释放和排卵 (FARMER & PAPCOFF 1979, BERGFELD & HARING 1983) 16

17 BIOLOGICAL ACTIVE SUBSTANCE TO STMULATE FOLLICLE GROWTH ecg + hcg (PG 600) 促进卵泡发育的药物 Combination of different doses of equine (ecg) and human chorion gonadotropin (hcg), mainly of 400 IU ecg and 200 IU hcg. 两种激素的复方制剂, 主要是 400IU 的 ecgi 和 200IU 的 hcg Disadvantage: too high amount of LH (hcg) activity; can cause premature luteinization of follicles and cysts 缺点 :LH 活性太高, 引起卵泡过早黄体化和囊肿 (SCHILLING & CERNE 1972, CHRISTENSON & TEAGUE 1975, BERGFELD et al. 1982, ESTIENNE et al. 2000, KNOX et al. 2001) 17

18 TIME AND DOSAGE OF ecg APPLICATION ecg 使用的时间和剂量 GILTS 后备母猪 TIME DOSAGE REFERENCE 24h after Regumate 烯丙孕素停药后 24 小时 48h after Regumate 烯丙孕素停药后 48 小时 IU BERGFELD et al IU POLGE et al BRÜ SSOW & BERGFELD IU CASSAR et al SOWS 经产母猪 24h after weaning 断奶后 24 小时 IU BERGFELD et al h after weaning 800 IU HÜ HN et al h after weaning * 1000 IU * BARBE et al h after weaning 800 IU SCHNURRBUSCH et al at weaning IU CASSAR et al * 1 st litter sows 初产母猪 18

19 Important prerequisites for fixed-time insemination are: 定时输精的重要先决条件 : all females would have a similar timed LH surge 所有母猪 LH 峰时间类似 all follicles would ovulate in a defined time window 所有母猪集中排卵 19

20 BIOLOGICAL ACTIVE SUBSTANCES TO ENSURE TIMED OVULATION 确保定时排卵的药物 hcg human Chorion Gonadotropin 人绒毛膜促性腺激素 high purified serum preparation from the urine of pregnant women 孕妇尿中高纯度的提取物 acts like LH on gonads 在性腺作用类似 LH stimulates final follicle maturation and subsequent ovulation 刺激卵泡最终成熟和后续排卵 HYPOTHALAMUS GnRH PITUITARY FSH LH GONADS ESTRADIOL / PROGESTERON plh porcine Luteinizing Hormone 猪促黄体素 biological preparation from porcine pituitary 猪脑垂体提取物 acts on gonads 作用于性腺 stimulates final follicle maturation and subsequent ovulation 刺激卵泡最终成熟和后续排卵 20

21 BIOLOGICAL ACTIVE SUBSTANCES TO ENSURE TIMED OVULATION 确保定时排卵的药物 GnRH Gonadotropin-Releasing Hormone 促性腺激素释放激素 synthetic derivatives of natural GnRH (deca- or nonapeptides) 合成的十肽 GnRH stimulates LH release and subsequent ovulation 促进 LH 释放和后续排卵 HYPOTHALAMUS GnRH PITUITARY FSH GONADS LH ESTRADIOL / PROGESTERON LH (ng/ml) GnRH Hours after GnRH Animal No. TIMED LH SURGE AND OVULATION AFTER GnRH APPLICATION GnRH 处理后 LH 峰和排卵时间 Days after Regumate 21

22 TIME AND DOSAGE OF hcg, plh AND GnRH APPLICATION hcg plh 和 GnRH 使用时间和剂量 DRUG TIME DOSAGE REFERENCE GILTS hcg 78-80h after ecg 500IU BERGFELD et al BRÜ SSOW et al GnRH 80h after ecg 50µg (1) BRÜ SSOW & BERGFELD 1979 BRÜ SSOW et al. 1996, h after ecg 10-20µg (2) BRÜ SSOW et al h after Regumate 6-16µg (3) DRIANCOURT et al SOWS hcg 72-74h after ecg 500IU BRÜ SSOW et al h after ecg 500IU HÜ HN et al plh 80h after ecg 5mg CASSAR et al at estrus onset 5mg FONTANA et al at standing heat 5mg ZAK et al GnRH 56-58h after ecg 50µg HÜ HN et al BRÜ SSOW et al h after weaning 6-16µg (3) DRIANCOURT et al h after weaning µg (4) KNOX et al FRANCISCO et al (1) GONAVET, (2) GOSERELIN, (3 ) RECEPTAL, (4) OVUGEL 22

TIME RELATION ESTRUS OVULATION 发情 - 排卵相对时间 SOWS ESTRUS DURATION 24 76 h 发情期间 24-76h 24 h 48

23 TIME RELATION ESTRUS OVULATION 发情 - 排卵相对时间 SOWS ESTRUS DURATION h 发情期间 24-76h 24 h 48 h 72 h E2 LH E2 LH GILTS ESTRUS DURATION h 发情期间 55-65h 24 h 48 h ESTRUS HEAT E2 LH 23

24 TIME OF OVULATION AFTER OVULATION INDUCTION 诱导排卵后的排卵时间 BRÜ SSOW et al BRÜ SSOW et al BRÜ SSOW et al CASSAR et al DRIANCOURT et al HUNTER 1976 KRAELING & WEBEL 2015 BERGFELD et al HOURS AFTER OVULATION INDUCTION 诱导排卵后的时间 hcg plh GnRH 24

25 Since ovulation period within a batch of sows can widely varying (32-48h after LH release or LH surge induction), the insemination should be selected in such way that a mature cohort of sperm will be present in the oviduct at ovulation. 因为一批母猪排卵时间不同 (LH 释放或者 LH 峰诱导后 32-48h), 所以输精应选择的方式是在排卵时输卵管中存在大量的成熟精子 25

26 Some introducing notes to understand sperm transport within the genital tract at ovulation in pigs 理解猪排卵时精子在生殖道运输的几个要点 : 26

27 (1) (2) (3) SPERM TRANSPORT IN THE GENITAL TRACT 精子在生殖道的传输 rapid passage through the uterus 快速通过子宫 colonization of a sperm reservoir in the oviduct 输卵管精子汇集 slow release towards the site of fertilization (AIJ) after ovulation 排卵后向受精点缓慢释放 AIJ CAUDAL ISTHMUS 峡部 UTERUS 子宫 27

OVUM TRANSPORT AND FERTILIZATION 卵子运输和受精 rapid transport of oocytes (30-45 min after ovulation) to the site of fertilization (AIJ ) 卵子快速 ( 排卵后 30-45min)

28 OVUM TRANSPORT AND FERTILIZATION 卵子运输和受精 rapid transport of oocytes (30-45 min after ovulation) to the site of fertilization (AIJ ) 卵子快速 ( 排卵后 30-45min) 传输到受精位点 release and transport of spermatozoa from the functional reservoir 功能性汇集的精子释放和传输 fertilization of oocytes each by one spermatozoa 每个卵子和一个精子结合受精 28

29 TIME REFERENCE OF FERTILIZATION EVENTS 受精事件中的参考时间 6h 2-12h 8-12h Ovulation period 排卵期 Oocyte life span 卵子寿命 Sperm capacitation 精子获能时间 - 24h Life span of sperm 精子寿命 HOURS (HUNTER 1974, BRÜ SSOW et al. 1990, 2007, WABERSKI et al. 1994, SOEDE et al. 1994) 29

30 INSEMINATION RELATIVELY TO FERTILIZATION EVENTS 受精事件中相对应的人工授精 1 st AI 2 nd AI OOCYTE LIFE SPAN 卵母细胞寿命 SPERM LIFE SPAN 精子寿命 OVULATION PERIOD 排卵阶段 HOURS AFTER OVULATION INDUCTION 诱导排卵后的时间 30

31 FIXED-TIME INSEMINATION PROTOCOL 定时输精程序 >4 WEEKS LACT. SOWS 经产母猪 < 4 WEEKS LACT. WEANING 断奶 24 h ecg h h hcg/gnrh hcg/gnrh GILTS 后备母猪 REGUMATE 停喂 24 h ecg h hcg/gnrh 1 st AI h 1 st AI h 2 nd AI h 2 nd AI h 31

32 FIXED-TIME INSEMINATION PROTOCOL WITHOUT ecg 无 ecg 的定时输精程序 SOWS 经产母猪 GILTS 后备母猪 >4 WEEKS LACT. < 4 WEEKS LACT. WEANING 断奶 REGUMATE 烯丙孕素 h h hcg/gnrh hcg/gnrh h hcg/gnrh 1 st AI h 1 st AI h 2 nd AI h 2 nd AI h 32

TIME TABLE OF ORGANISATION OF FIXED-TIME INSEMINATTION >4 WEEKS LACTATION > 哺乳期 4 周 SOWS 经产母猪 <4 WEEKS LACTATION 哺乳期 <4 周 GILTS 后备母猪 WEDNESDAY 周三 THURSDAY 周四 FRIDAY 周五 SATURDAY 周六 SUNDAY 周日 MONDAY 周一

33 TIME TABLE OF ORGANISATION OF FIXED-TIME INSEMINATTION >4 WEEKS LACTATION > 哺乳期 4 周 SOWS 经产母猪 <4 WEEKS LACTATION 哺乳期 <4 周 GILTS 后备母猪 WEDNESDAY 周三 THURSDAY 周四 FRIDAY 周五 SATURDAY 周六 SUNDAY 周日 MONDAY 周一 TUESDAY 周二 am pm am pm am pm am pm am pm am pm am pm last Regumate WEANING 24 h WEANING 24 h ecg 24 h ecg ecg h h h GnRH or hcg GnRH or hcg GnRH or hcg h h h 1 st AI 1 st AI 1 st AI 2 nd AI h 2 nd AI h 2 nd AI h 33

34 NOVEL ASPECTS OF FIXED-TIME INSEMINATION 定时输精创新 Usually, insemination occurs intravaginal with 2-3x10 9 sperm in ml extender (KIRKWOOD & KAUFFOLD 2015). 通常, 阴道内输精 ml 混合精液, 含有个精子 Transcervical deep intrauterine insemination can reduce the sperm concentration to 10x10 6 sperm when inseminated close to the utero-tubal junction (KRÜ GER & RATH 2000, MARTINEZ et al. 2001, ROCA et al. 2003, BRÜ SSOW et al. 2013). 子宫内深部输精, 输精位点在子宫输卵管结合部可降低到 10x10 6 精子 34

35 However, the timing of insemination relative to ovulation becomes more important as the number of sperm deposited is reduced (ROZEBOOM et al. 2004) or single fixed-time insemination will be done (BRÜ SSOW et al. 2013, DRIANCOURT 2013). 而且, 基于排卵时间的输精, 比减少精子数量更重要, 或者一次性定时输精将来可以做到 Therefore, timed ovulation induction is the ultimate goal of successful single fixed-time insemination. 因此, 诱导定时排卵是一次性定时输精成功的最终目标 35

36 DEVICES FOR INTRAUTERINE AND DEEP INTRAUTERINE INSEMINATION 子宫内输精和子宫内深部输精装置 (MARTINEZ et al. 2001, BELSTRA et al. 2003) 36

SUMMARIZING REMARKS 总结 Fixed-time insemination in a batch of sows requires synchronized follicle maturation, timed onset of estrus and ovulation, and the presence of fertile sperm close to ovulation.

37 SUMMARIZING REMARKS 总结 Fixed-time insemination in a batch of sows requires synchronized follicle maturation, timed onset of estrus and ovulation, and the presence of fertile sperm close to ovulation. 一个批次的母猪定时输精需要卵泡成熟同步化, 定时发情和排卵, 排卵时有精子存在 There exists a profound knowledge regarding the physiology of reproduction in the pig and the impact of hormones and hormonal substances to regulate ovulation and fertilization processes. 关于猪的生殖生理激素或者激素样物质对排卵和受精过程的影响等知识要有深刻的认识 37

38 SUMMARIZING REMARKS Biological active compounds (as ecg, hcg, GnRH) which stimulate and regulate the reproductive processes in the pig are available. Their proper application considers the physiological specifics of porcine reproduction and enables fixed-time insemination. 生物活性物质 ( 如 ecg, hcg, GnRH ), 刺激和调节猪的生殖过程, 需要使用它们的适当使用顾及到猪生殖过程中的生理细节, 能够定时输精 Strict application of fixed-timed insemination procedures will ensure fertility, rationalize reproduction and improve reproductive outcome. 严格按照定时输精程序执行, 确保生殖, 合理繁殖, 提高繁殖效果 38

39 THANKS FOR YOUR ATTENTION 感谢您的关注 39

2 3 4 5 22 22 23 25 35 38 39 45 46 1

97017626 O O 2 3 4 5 22 22 23 25 35 38 39 45 46 1 intrauterine insemination IUI 202 IUI a 24 IUI b 24 IUI c hcg 36 IUI d hcg 24 48 IUI 202 262 30 2 1 27 1 14.9% 11.5% a 122 151 13.9%, 11.3% b 42 46 2.38%,