Distribution of Live and Dead Spermatozoa in the Genital Tract of Gilts at Different Times After Insemination

Twenty-four gilts were inseminated pair-wise with live or dead spermatozoa from the same ejaculate. The insemination dose was 100 ml undiluted semen containing, on average, 19×10⁹ spermatozoa. The gilts were slaughtered 1, 2, 6 and 12 h after insemination. The numbers of spermatozoa were counted in the uterus, uterotubal junction and in four equally long segments of the oviduct, called I–IV, with a haemocytometer. IV was adjacent to the uterotubal junction. The numbers of spermatozoa recovered in the uterus diminished significantly during the first 12 h after insemination. From gilts inseminated with live spermatozoa more spermatozoa were recovered in the uterotubal junction than from gilts inseminated with dead spermatozoa. Two h after insemination spermatozoa were recovered in all oviducts. Significantly more live than dead spermatozoa were recovered in Segments III and IV of the oviduct, regardless of time. In gilts inseminated with live spermatozoa the sperm count in Segment I varied significantly with time, being hiigest 2 h after insemination. At 6 and 12 h there were no distinct differences in the distribution of live spermatozoa between the various oviduct segments. The numbers of spermatozoa recovered in the oviduct were at these times apparently related to the sperm depots in the uterotubal junction.     

母牦牛的繁殖特性与人工授精

牦牛是青藏高原特有的遗传资源,是高寒牧区的基本生产生活资料。牦牛繁殖是牦牛生产中的关键环节,深入了解母牦牛的繁殖特性是实施牦牛繁殖调控的基础。本文从初情期、性成熟、发情、妊娠期、分娩、繁殖力等方面综述了牦牛的繁殖特性,并探析了提高牦牛人工授精效率的技术措施,旨在为牦牛品种改良提供技术参考。     

猪深部输精技术应用研究进展

常规子宫颈人工授精(cervical artificial insemination,CAI)技术输精量大,存在浪费和不经济的问题,尤其在使用高附加值的精液产品时更为突出。近年来,一种高效利用猪精液的深部输精技术得到了研究和应用,这种技术与定时输精技术相结合,直接将精液输送至子宫体、子宫角和输卵管内,减少了每头母猪受孕所需精子数目,大幅度提高了良种公猪的利用价值,有助于实现冷冻保存精液和性控精液在养猪生产上的商业化应用。作者概述了猪人工授精中3种深部输精技术在液态保存、冷冻保存和性控精液上的研究和应用情况。无论实施哪种输精技术,公猪繁殖力、精子活力、母猪的饲养管理、输精时间和人工授精技术操作的规范性都是提高受胎率必须要考虑的因素。     

Sperm Storage in the Female Reproductive Tract in Birds

The ability to store sperm in the female genital tract is frequently observed in vertebrates as well as in invertebrates. Because of the presence of a system that maintains the ejaculated sperm alive in the female reproductive tract in a variety of animals, this strategy appears to be advantageous for animal reproduction. Although the occurrence and physiological reasons for sperm storage have been reported extensively in many species, the mechanism of sperm storage in the female reproductive tract has been poorly understood until recently. In avian species, the specialized simple tubular invaginations referred to as sperm storage tubules (SSTs) are found in the oviduct as a sperm storage organ. In this review, we summarize the current understanding of the mechanism of sperm uptake into the SSTs, maintenance within it, and controlled release of the sperm from the SSTs. Since sperm storage in avian species occurs at high body temperatures (i.e., 41 C), elucidation of the mechanism for sperm storage may lead to the development of new strategies for sperm preservation at ambient temperatures, and these could be used in a myriad of applications in the field of reproduction.     

Incidence of Unilateral Fertilizations after Low Dose Deep Intrauterine Insemination in Spontaneously Ovulating Sows under Field Conditions

A new procedure for non-surgical deep intrauterine insemination (DUI) in unrestrained sows hormonally induced to ovulate, has been reported. In comparison with standard artificial insemination (AI), with this procedure, the sperm numbers inseminated can be reduced 20-fold without reducing the reproductive performance of these hormonally treated sows. The present study evaluated, using two experiments, the reproductive performance applying 20-fold different sperm numbers per AI dose using DUI or standard AI in spontaneously ovulating sows, under field conditions. In experiment 1, AI was applied to crossbred sows at 12, 24 and 36 h after onset of spontaneous oestrus using one of the following two regimes: (i) DUI (treatment) with 0.15 x 10(9) fresh boar spermatozoa in 5 ml of Beltsville thawing solution (BTS) extender (n = 95), and (ii) standard cervical AI (control) with 2.85 x 10(9) fresh spermatozoa in 95 ml of BTS extender (n = 95). The farrowing rates of the two groups of sows were statistically similar (NS). However, a decrease (p < 0.002) in litter size and the total number of pigs born alive was observed in sows inseminated with the DUI procedure. In experiment 2, 42 post-weaned oestrus sows were inseminated following the same design described for experiment 1 during spontaneous oestrus. On day 6 after onset of oestrus, the proximal segment of the uterine horns of the sows were flushed under surgery to retrieve eventual embryos and evaluate the success of fertilization per cornua (e.g. occurrence of effective uni- vs bilateral sperm transport rendering uni- or bilateral, complete or partial fertilization). Retrieved embryos were assessed for cleavage and number of accessory spermatozoa. Although identical overall pregnancy rates were achieved in both insemination groups, the percentage of sows with partial bilateral fertilization and unilateral fertilization was markedly higher (p < 0.05) in the DUI group (35%) compared with the control (standard AI) group (5%), with a consequent lower (p < 0.001) percentage of viable early embryos after DUI. The number of accessory spermatozoa in the zona pellucida of the embryos was highly variable, but higher (p < 0.001) in control animals than in DUI-AI. No accessory spermatozoa were found in oocytes retrieved from sows depicting unilateral fertilization. In conclusion, DUI in spontaneously ovulating sows with 0.15 x 10(9) spermatozoa renders similar farrowing rates but a lower litter size compared with use of standard AI with a 20-fold higher sperm dose. The lower litter size ought to be related to a decreased distribution of spermatozoa after DUI leading to a higher incidence of partial bilateral and unilateral fertilization.     

Immunolocalization of the Cholesterol Transporters ABCA1 and ABCG1 in Canine Reproductive Tract Tissues and Spermatozoa

The mammalian sperm membrane undergoes cholesterol efflux during maturation and fertilization. Although ATP‐binding cassette (ABC) transporters are known to transport cholesterol through cell membranes in other organs, their presence in canine testis, epididymis and sperm has not been proven to date. Hence, the aim of the present study was to localize the ABC transporters ABCA1 and ABCG1 in canine testicular and epididymidal tissue as well as in spermatozoa membranes. To this end, semen samples from 12 dogs as well as testicles and epididymides of four young and healthy dogs were prepared for immunohistochemistry, respectively. Capacitation and acrosome reaction (AR) were induced in aliquots of the semen samples before immunostaining to assess changes in the expression of ABCA1 and ABCG1. Evaluation by confocal microscopy revealed the presence of both ABCA1 and ABCG1 in canine testicles and of ABCA1 in the epididymides. In spermatozoa, only ABCA1 immunoreactivity was detected, mainly in the region of the acrosome and midpiece. After induction of capacitation, ABCA1 signal persisted in the acrosome but disappeared after AR, indicating a loss of ABCA1 with the loss of the acrosome. We conclude that ABCA1 and ABCG1 are expressed in canine testis, whereas only ABCA1 is expressed in epididymis and spermatozoa membrane, both transporters probably contributing to the regulation of membrane cholesterol content.     

Distribution and Heterogeneity of Mast Cells in Female Reproductive Tract and Ovary on Different Days of the Oestrus Cycle in Angora Goats

The physiological distribution of mast cells (MCs) in the reproductive tract and ovary of 12 Angora goats was determined using light microscopic histochemical techniques. Uterus (corpus uteri and cornu uteri), uterine cervix, uterine tubes (isthmus and ampulla) and ovary samples were obtained by laparatomy from groups of animals during metoestrus, dioestrus and proestrus (days 5, 10 and 16 of the oestrous cycle). Tissues were fixed in Mota's fixative (basic lead acetate) for 48 h and embedded in paraffin. Six-micrometre-thick sections were stained with toluidine blue in 1% aqueous solution at pH 1.0 for 5 min and alcian blue-Safranin at pH 1.0 for 30 min. MCs were generally associated with blood vessels in all reproductive organs. In the uterus, they were concentrated mainly in the close of the uterine gland and deep stroma in the endometrium. Higher MC numbers were observed by toluidine blue staining in the uterus, uterine cervix and uterine tubes on days 10 (corpus uterine: 4.7 ± 3.8 and cornu uterine: 4.9 ± 3.5) and 16 (corpus uterine: 5.9 ± 4.5 and cornu uterine: 5.4 ± 2.4) of the oestrous cycle compared with day 5 (p < 0.05). Mast cells were not observed in the follicles, the corpus luteum and the underside of the surface epithelium of the ovarian cortex, but were observed in the interstitial cortical stroma and the ovarian medulla. In the ovary, MC numbers were significantly higher on day 16 of the oestrous cycle (cortex: 3.4 ± 2.4 and medulla: 5.7 ± 4.5, p < 0.05). Safranin-positive connective tissue MCs were not observed in the uterine tube on any occasion. These results indicate oestrous cycle-related changes in the number and location of MCs in goat reproductive organs.     

雌性生殖道内神经支配及相关功能探讨

本文概述了近年来有关神经在雌性生殖道内分布的研究概况，主要是子宫内神经分布及其传入神经分布，并阐述了在妊娠期内胆碱能神经和肾上腺素能神经分布的变化；探讨了生殖道内神经与子宫运动及子宫免疫的关系。     

The Reproductive Tract of the Porcine Female (A Biometrical Study)

A biometrical study of female porcine reproductive tracts recovered after slaughter was carried out. Sizes and weights of the various areas from one hundred eighty-seven sow tracts, eighty-one pre-puberal gilts and thirty-two puberal gilts were recorded. A table of abnormalities observed in examination of the sow tracts is also presented.     

Influence of Boar Seminal Plasma on the Distribution of Spermatozoa in the Genital Tract of Gilts

The main purpose of the present study was to investigate whether boar seminal plasma affects the transport of spermatozoa in the genital tract of oestrous pigs or not, with special reference to the sperm transport into the oviducts. Altogether 17 gilts were used in three experiments.Experiment I. In nine gilts one uterine horn was injected surgically with 10¹⁰ spermatozoa suspended in seminal plasma and the other uterine horn with 10¹⁰ spermatozoa suspended in TESNaK-glucose buffer solution. The sperm deposition was performed under general anaesthesia. The gilts were slaughtered 1–2 or 4–6 h after insemination. The genital tract was removed and the numbers of spermatozoa determined in oviducts and in uterine horns.Experiment II. The insemination doses were prepared exactly as in Experiment I. Approx. 24 h before insemination Polyvinylchloride cannulas were inserted into the uterine lumen of the horns, drawn via the midventral incision at linea alba subcutaneously to cutaneous incisions ventral to the vulva opening. One cannula was placed in each uterine horn. At standing heat the insemination doses were slowly injected through the cannulas. The gilts were slaughtered 1 h after insemination and the numbers of spermatozoa within the genital tract were counted.Experiment III. In three gilts under general anaesthesia the uterine horns were ligated 10 cm from the uterotubal junction. The semen doses (containing 2 × 10⁹ spermatozoa), prepared as in Experiment I, were deposited into the uterine horns anterior to the ligatures through a cannula. The gilts were slaughtered 1 h after insemination, and the numbers of spermatozoa within the oviducts and ligated part of the uterine horns were counted.In all three experiments more spermatozoa were, on average, recovered in the oviducts connected to uterine horns inseminated with spermatozoa suspended in seminal plasma. In Experiments I andII this was the case for 10 of 14 gilts and in Experiment III for all the three gilts. It is therefore suggested that boar seminal plasma pro¬motes sperm transport into the oviduct of oestrous pigs. The back¬ground mechanism for this is discussed.     

The Role of Platelet‐activating Factor in the Mammalian Female Reproductive Tract

Platelet‐activating factor (PAF) is a potent lipid mediator produced by various cell types of mammals and is involved in an inflammatory‐like process with increased vascular permeability. Platelet‐activating factor exerts its actions through the activation of specific PAF receptors (PAF‐R) found in cells and tissues of the female reproductive tract. The aim of this article was summarized briefly in the current research on the role of PAF in female reproductive functions. Platelet‐activating factor has been implicated in processes of ovulation, implantation and parturition because of its angiogenic and growth factor properties. This factor is influenced by ovarian steroid hormones in bringing about changes in the uterus and is a candidate molecule for initial embryo–maternal dialogue. Tissue concentrations of PAF are regulated by the equilibrium between biosynthesis and degradation by PAF‐acetylhydrolase (PAF‐AH). Antagonists of PAF interfere with ovulation and implantation. Platelet‐activating factor, its receptor, and PAF‐AH activity play an important role in the maintenance of pregnancy.     

促黄体素受体在母畜生殖道的分布与表达

在多种动物包括灵长类动物、猪、牛、火鸡和人,子宫内都发现有促黄体素(LH)或人绒毛膜促性腺激素(hCG)及其mRNA的结合位点.牛、猪在整个发情周期子宫内都存在LH受体(LHR)蛋白及其mRNA,并在黄体期表达最高.LH通过激活腺苷酸环化酶和磷脂酶C信号途径,增加前列腺素合成酶(PGSH),也称环加氧酶(COX)的活性,从而促进前列腺素(PG)的合成.牛、猪、羊在发情周期的15 d左右PGF2α升高,这与子宫内LHR的活化有关.在不同种动物的输卵管、子宫内膜、子宫肌层和子宫颈,LHR的释放呈现出动态特征,表明LH在发情周期和胚胎附植的分子自分泌-旁分泌调节中可能具有重要的作用.     

Identification of the DDAH2 Protein in Pig Reproductive Tract Mucus: A Putative Oestrus Detection Marker

Precisely detecting oestrus is important for artificial insemination. The aims of this study were to identify oestrus‐specific sow mucus proteins to determine the optimal time for artificial insemination. The proestrous‐ and oestrous‐stage mucus proteins were purified and analysed with proteomic tools such as two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization–time‐of‐flight analyses. Among the differentially expressed proteins, the dimethylarginine dimethylaminohydrolase 2 (DDAH2) protein showed a 3.6‐fold increase during the proestrous stage compared to that during the oestrous stage. A western immunoblot study revealed that two of three sow mucus samples clearly showed negative anti‐DDAH2 antibody activity during the oestrous stage. This study demonstrated that the pig DDAH2 mucus protein exists during the proestrous stage, but not during the oestrous stage, suggesting that mucus DDAH2 could be useful as an oestrus detection marker.     

防御素在雌性哺乳动物生殖系统中的分布和功能研究进展

防御素(defensin)作为抗菌肽的主要家族,在哺乳动物体内广泛分布,主要表达于中性粒细胞和上皮细胞,具有广谱的抗菌活性和多方面免疫调节功能.近年来,发现防御素广泛在哺乳动物雌性生殖系统中表达,发挥抑菌抗炎的作用,预防微生物对子宫或生殖道等的感染,从而维持雌性生殖系统的环境稳定,同时参与动物生殖.论文结合已有的研究成...     

Development after Separation and Reaggregation of Blastomeres from Early Pig Embryos cultured in vitro

The in vitro developmental potential of blastomeres from early porcine embryos war studied. Two-, 4-, and 8-cell embryos were separated into component blastomeres and replaced into zonae pellucidae in the following configurations (designated by number of blastomeresl original cell stage): half embryos (1/2, 2/4, and 4/8); quarter embryos (1/4 and 2/8); and eighth embryos (1/8). Culture was done in 50 μl droplets under paraffin oil in either Whitten's medium supplemented with 4 mg/ml bovine serum albumin (BSA) and 10% fetal calf serum (FCS) or in the same medium supplemented with porcine oviductal cells. Cultures were observed on a daily baris when the morphology was recorded and at the end of culture (144—168 h after the expected time of ovulation), the embryos were stained with Hoechst 33342 and cell counts made. Generally, no significant differences in developmental rate were found between the two culture system and the data was pooled. Blastocyst formation rate in half embryos was 23%, 28%, and 44% for 1/2, 2/4, and 4/8 embryos, respectively. The respective blastocysts contained an average of 24.3 ± 4.4 and 28.8 ± 2.0 versa 41.2 ± 6.4 cells (p < 0.05 and p < 0.05). Blastocyst formation rate in quarter embryos was 15% and 35% (p < 0.01) for 1/4 and 2/8 embryos, respectively and they possessed an average of 17.0 ± 1.8 and 22.8 ± 1.4 cells (p < 0.02), respectively. Blastocyst formation rate in 1/8 embryos was 9% and the blastocysts averaged 13.0 ± 1.3 cells. Two-,4-, and 8-cell control embryos formed blastocysts at rates of 22%, 85%, and 64%, respectively, and had average cell counts of 45.8 ± 10.8, 69.1 ± 4.0, and 75.4 ± 7.3, respectively. The timing of the initiation of blastocoel formation was similar for reaggregated and control embryos that started culture at the same stage. This along with the cell counts of reaggregated embryos at cavitation, which were proportional to the reduction in cell mars (7.2 ± 0.6, 12.6 ± 1.9, and 25.6 ± 2.8 for quarter, half and control embryos, respectively) supports the assumption that in pigs blastocoel formation occurs independent of the number of cells present. Inhalt: Entwicklung von frühen in vitro kultivierten Schweineblastomeren nach Abtrennung und Reaggregation Es wurde die Entwicklungspotenz von Blastomoren aus frühen Schweineembryonen studiert. Zwei-, Vier- und Achtzell-Embryonen wurden in ihre Blastomerenbestandteile zerlegt und nachfolgendem Schema in Zonae pellucidae zurückverbracht: Halbierte Embryonen (1/2, 2/4, 4/8) Viertelembrynnen (1/4 und 2/8) und Achtelembryonen (1/8). Die Kulturphase erfolgte in 50 μl Tropfen, die mit Parafinöl überschichtet waren. Es wurde Whitten's Medium, entweder mit 4 mg/ml BSA und 10% FCS verwendet oder das Medium mit porcinen Eileitenellen supplementiert. Die Kultur wurde täglich überpüft und die Morphologie beurteilt. Am Ende der Kulturzeit (144–168 Std nach dem berechneten Ovulationszeitpunkt) wurden die Embryonen mit dem Hoechstfarbstoff 33342 gefärbt und die Zellen gezählt. Signifikante Unterschiede in der Entwicklungsrate zwischen den beiden Medien wurden nicht gefunden und die Daten wurden zusammengefaβt. Die Blastozystenbildungsrate der Embryonenhälften betrug 23% und 44% für 1/2, 2/4 und 4/8 Embryonen. Diese Blastozysten enthielten im Mittel 24,3 ± 4,4 und 28 ± 2,0 gegenüber 41,2 ± 6,4 Zellen (p ≤ 0,05). Die Blastozystenbildungsrate aus Embryoversuchen betrug 1.5% und 35% (p ≤ 0,01) für 1/4 und 2/8 Embryonen. Im Mittel waren 17,0 ± 1,8 und 22,8 ± 1,4 Zellen (p ≤ 0,02) enhvickelt worden. Die Blastozystenbildungsrate in 1/8 Embryonen betrug 9% und durchschnittlich wurden 13,0 ± 1,3 Zellen in den gebildeten Blastozysten nachgewiesen. Zwei-, Vier- und Achtzell-Embryonen der Kontrollgruppe formten zu 22%, 85% und 64% Blastozysten und die mittlere Zellzahl betrug 45,8 ± 10,8, 69,1 ± 4,0 und 75,4 ± 7,3. Der Zeitpunkt der Blastozoelbildung war für reaggregierte und Kontrollembryonen ähnlich, wenn sie die Kulturphase im gleichen Enhvicklungsstadium begannen. Diese Tatsache und die vorhandene Zellzahl in reaggregierten Embryonen zum Zeitpunkt der Kavitation, die sich proportional zur Verringerung der Zellzaiilen verhielt (7,2 ± 0,6, 12,6 ± 1,9 und 25,6 ± 2,8 für Viertel-, Halb- und Kontrollembryonen), unterstützt die Annahme, daβ die Blastozoelbildung beim Schwein unabhängig yon der Zellzahl erfolgt.     

定时输精对经产母猪繁殖性能及生殖激素的影响

试验旨在建立高效的经产母猪定时输精（timed artificial insemination,TAI）技术,研究了定时输精对经产母猪繁殖性能、断奶-分娩间隔、不同胎次母猪产仔性能及断奶后7 d内血清生殖激素水平的影响。选取309头2~8胎次二元（长×大）经产母猪,随机分为对照组和试验组,对照组母猪进行常规人工授精（artificial insemination,AI）,试验组母猪进行断奶后24 h注射PMSG 1 000 IU,间隔72 h注射GnRH 100 μg,在注射GnRH后24和40 h各输精1次的定时输精技术。通过统计两组母猪的断奶1周内发情率、受胎率、分娩率、窝均产仔数等,判断定时输精对经产母猪繁殖性能的影响;通过对断奶时间和分娩时间的统计,检测定时输精对经产母猪断奶-分娩间隔的影响;用放射免疫（RIA）方法检测2~4胎次母猪断奶1周内血清E₂、LH、FSH和P₄的含量,研究定时输精对母猪生殖激素的影响。结果显示,试验组母猪发情率显著高于对照组（P<0.05）,但两组间受胎率、分娩率差异不显著（P>0.05）,窝均产仔数、窝均合格仔数和繁殖效率有增加的趋势,但差异不显著（P>0.05）;定时输精显著缩短了母猪的断奶-分娩间隔（P<0.05）。在胎次方面,3~4胎母猪使用定时输精的效果较好,其发情率、受胎率和分娩率均显著高于对照组（P<0.05）。在生殖激素方面,试验组E₂水平在注射PMSG后迅速上升,且在定时输精处理后66~96 h内持续高于对照组（P<0.05）,试验组P₄水平在断奶后至配种前显著低于对照组（P<0.05）,但配种后快速升高,并高于对照组;LH和FSH的含量在两组间无显著差异。综上,定时输精可有效提高经产母猪的断奶发情率,并减少其非生产天数,可显著提高3~4胎母猪的繁殖性能。     

Morphological and Morphometric Description of Female Reproductive Tract of Sapajus apella (Capuchin monkey)

Forest destruction has progressively hampered the survival of many species, and this is why it is so important to study of the lives of primates in captivity. This study aimed to describe the morphological aspects of the female reproductive tract of Sapajus apella. We used five animals obtained from the National Primate Center, Ananindeua – PA. The ovaries were paired, compact and symmetrical and had a smooth surface. The uterine tubes were bilateral and convoluted in adult animals and straight in young individuals. The uterus was simple and located in the pelvic region. The vagina was a long structure due to the position of the uterus. The external genitalia were located in the urogenital perineum and consisted of dark pigmented labia majora and labia minora, a vaginal vestibule as long as the vagina and a well‐developed clitoris. The results showed that the genitals of S. apella resemble those of other Neotropical primates.     

Insemination with Sex Sorted Fresh Bovine Spermatozoa Processed in the Presence of Antioxidative Substances

Flow cytometrically sex sorted spermatozoa are reduced in their fertilizing capacity, particularly when stored either in cooling extender or after freezing in liquid nitrogen. So far, preservation methods for sorted spermatozoa have differed only marginally from procedures used for unsorted semen. In the present study, a TRIS extender was modified to balance major cell damage caused by the sorting process and by liquid storage of the sorted spermatozoa. The new extender, containing a combination of antioxidants (AO) and bovine serum albumin (BSA), significantly increased the lifespan and fertilizing capacity of sex sorted spermatozoa. No significant differences were observed between unsorted controls and sorted samples for motility and status of sperm membranes as tested by fluorescein-isothiocyanat-peanut agglutinin/propidium iodide (FITC-PNA/PI). Acrosome integrity of spermatozoa was significantly better when semen was stored at 15 degrees C for 24 and 48 h in an extender containing AO with or without BSA as compared with controls (p < 0.05). There were no significant differences, in pregnancy rates of heifers inseminated at a natural oestrus, between unsorted controls (16/24, 66.7%) and both sorted groups (AO + BSA: 18/31, 58.1% and AO-BSA: 12/22, 54.5%). Additionally, it was shown for the first time that artificial insemination (AI) with liquid sexed bull spermatozoa stored for 72 h after sorting can result in pregnancy rates similar to AI with non-sorted semen.     

引进母猪繁殖性能和仔猪生长性能的对比试验

在11个月的试验期内,对加系长白和英系大约克两品种间进行四种选配方式的纯繁和杂交,结果为长白(♂)×约克(♀)的杂交选配方式最优。用长白做父本的组合方式在纯繁和杂交中的繁殖性能及指标都高于约克做父本的组合方式。