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Maturation, Fertilization, and Development of Dog Oocytes in Vitro1
Abstract
Preovulatory oocytes were collected from ovaries of beagle bitches that had received superovulatory treatment. They were cultured in a modified Krebs-Ringer bicarbonate solution containing 10% fetal calf serum and 30 mg/L gentamicin sulfate for up to 72 h. About 32% of oocytes reached metaphase II by 72 h of culture. When these in vitro-matured oocytes were inseminated with ejaculated beagle spermatozoa that had been preincubated for 4 h, sperm penetration of the zona pellucida started about 1 h after insemination, and both male and female pronuclei were seen in the ooplasm at 8 h after insemination. At 18-20 h after insemination, oocytes were transferred to Whitten's medium and cultured for 76-78 h. The first cleavage was observed at 48 h after insemination, and 15 of 45 oocytes developed to the 2-8-cell stage. These results demonstrate that in vitro-matured canine oocytes can be fertilized and develop to the 8-cell stage in vitro.
... Petri kabında toplanan sıvı bir stereo-mikroskop altında (15x büyütmede) incelenerek oositler araştırılır. 13,14 Kültür için koyu pigmentli sitoplazmaya sahip bir veya daha fazla kumulus hücre tabakası ile çevrelenmiş primer oositler seçilir. [15][16][17][18] Primer oositler elde edildikten sonra 72 saat içerisinde konservasyonları yapılmış, 48 saatlik bir inkübasyon süresinden sonra yapılan incelemede bir olgunlaşma olmadığı gözlenmiştir. ...
... Buna karşılık, süperovulasyondan sonra %32' ye varan bir olgunlaşma oranı ve proöstrüstaki oositlerde ise %67' ye kadar varan bir olgunlaşma elde edilebilmiştir. 13,14,21 Siklus döneminin yanısıra, olgunlaşma ortamı da yumurta olgunlaşma oranını etkilemektedir. [18][19][20] Olgunlaşma ortamındaki östradiolün 1 ug/ml'den 20 μg/ml'ye yükseltilmesi ve insan somatotropininin eklenmesi ile olgunlaşma oranı %10,2'den %14,1'e yükselmiştir. ...
... [18][19][20][21]24,25 Bu amaçla %5 ve %10 oranında fötal buzağı serumu ilaveli doku kültürü solüsyonları tercih edilmiştir. 13,18 Bazı İsmail Kırşan ve ark. ...
Köpeklerde yardımcı üreme teknikleri (YÜT), diğer evcil hayvanlardan daha az gelişmiştir. Türe özgü üreme özellikleri, yani monoöstrik, poliovulatör ve mev-simsel olmayan üreme döngüsü, köpeklerde YÜT ile üremenin zorluk derece-sini arttırmakta ve başarı seviyesini düşürmektir. Metafaz II aşamasında olgun oositleri yumurtlayan çoğu memelinin aksine, köpekler germinal vezikül aşamasında olgunlaş-mamış oositleri yumurtlar ve oositler yumurtalıkta 48-72 saatlik bir postovulatör ol-gunlaşma dönemine girerler. Köpek oositlerinin in vitro olgunlaşması (İVO) için uygun bir sistem kurulmasına odaklanan birçok çalışmaya rağmen, verimlilik diğer memeli tür-lerinden daha düşüktür. 1,2 Üreme performanslarının iyileştirilmesi, yardımcı üreme teknolojilerinin (YÜT) nihai hedefidir ve bu amaç doğrultusunda bilimsel topluluklar tarafından biyolojik çe-şitliliğin korunmasına ilgi duyulmasıyla birlikte karnivorlarda da bu çalışmalar başlatıl-mıştır. Aslında, karnivorlar sadece evcil hayvan olarak değil, aynı zamanda evcil olmayan köpek ve kedi türlerine yönelik çalışmalar için karşılaştırmalı modeller olarak kabul edilir. Suni tohumlama (ST), in vitro fertilizasyon (IVF) ve embriyo üretimi (EÜ), embriyo transferi (ET) ve gamet dondurarak saklama (kriyoprezervasyon) gibi yardımcı üreme teknolojileri (YÜT), yalnızca biyolojik çeşitliliği korumak için değil, temel araş-tırma ve reprodüktif performansı geliştirme için de önemli biyoteknolojilerdir. Bunlar arasında, IVF-ET ve ET, potansiyel olarak döllenmeyi incelemek ve nadir türlerin ge-Köpeklerde Embriyo Elde Edilmesi ve Transferi Çalışmaları Collection of Embryo and Studies on Embryo Transfer in Dogs ÖZET Günümüzde memelilerin çoğaltılmasına yönelik yardımcı üreme teknikleri giderek artmaktadır. Ancak köpeklerde özellikle oositlerin elde edilmesi, in vitro olarak olgunlaştırılmaları, döllenmeleri ve elde edilen embriyoların olgunlaştırılması ve transferiyle ilgili biyoteknolojiler henüz tam oluşturulma-mıştır. Oysa diğer hayvan türlerinde, özellikle sığırlarda bu alanda biyoteknolojik yöntemler geliştiril-miş ve başarıyla uygulanmaktadır. Bu makalede köpeklerden oosit ve embriyo elde edilmesi, bunların olgunlaştırılmaları keza transfer yöntemleri ve neticede elde edilen başarı sonuçları hakkında ayrıntılı bil-giler verilmiştir. Anah tar Ke li me ler: Embriyo yapıları; köpekler; oosit; embriyo transferi ABS TRACT Nowadays, assisted reproductive techniques for reproduction of mammals are increasing. However, biotechnology related to obtaining oocytes, in vitro oosit maturation, fertilization, embryo maturation and transfer of the embryos obtained has not been established yet. Whereas, in other animal species, especially in cattles, biotechnological methods have been created and applied successfully in this field. In the presented article, detailed information about obtaining oocytes and embryos, their matura-tion, transfer methods and consequently success results in dogs are given.
... Dans ces espèces, un traitement de superovulation est en général administré à la femelle donneuse pour augmenter le nombre d'embryons à collecter. Cependant, la chienne ne répond pas ou mal aux traitements hormonaux de superovulation classiquement utilisés (eCG/hCG ; Archbald et al. 1990 ;Yamada et al. 1992). D'autre part, jusqu'à 9 jours après la fécondation, la collecte des embryons s'effectue par rinçage des oviductes et donc nécessairement par voie chirurgicale, donc invasive. ...
... La collecte des ovocytes peut s'effectuer par ponction de follicules préovulatoires (Yamada et al. 1992 ) (figure 3). Elle peut également se réaliser par section, à l'aide d'une lame de rasoir, des ovaires prélevés par ovariectomie (Reynaud et al. 2004) : cette modalité est celle qui est la plus couramment utilisée pour les études fondamentales menées sur l'ovocyte canin et sa maturation in vitro. ...
... La plupart (60 %) n'ont même pas repris leur méiose (Saint-Dizier et al. 2001 ;Luvoni et al. 2005). Avec des ovocytes issus de follicules préovulatoires, le taux de maturation final est de 32 % (Yamada et al. 1992). ...
There is very little data available on the specificities of oocyte and embryo biology in bitches. The main difference with other mammals is the time of meiosis resumption: it does not occur before ovulation, but in the oviduct 48 to 60 hours later The factors responsible for this delay are not known, which may explain why current in vitro maturation rates are so low (10 to 30%). Oocyte harvesting is also a major limiting factor, as there is no effective protocol for the induction of cycles and superovulation. In vitro fertilisation rates are equally low (10%), with a high rate of polyspermia. No puppy has yet been born from an embryo produced in vitro. As for embryos produced in vivo, their collection is difficult due to anatomical reasons and to the fact that superovulation cannot be induced. Embryo transfer to donor bitches is also hindered by difficulties to synchronise ovulations between donor and recipient bitches. Only 6 such trials have been reported in the literature, resulting in the birth of 45 puppies, In vitro cultures are very rarely used, and only four puppies were born from somatic cell cloning with only few hours of in vitro culture. Canine reproductive biotechnologies have thus largely fallen behind, due to a lack of fundamental research to improve our understanding of its specific physiological mechanisms. This deficit is all the more damaging that dogs are increasingly used as relevant biomedical models.
... Yamada i wsp. (29) wykonywali codzienne iniekcje, podając zwierzętom 100-400 µg estronu, aż do momentu pojawienia się wypływu z pochwy. Po trzech dniach, w formie iniekcji podskórnej, podano eCG w ilości 400 UI i hCG w ilości 1000 UI, a po kolejnych trzech i czterech dniach 10 µg estradiolu domięśniowo. ...
... Przykładowo: może to być pozbawiony dodatków PBS (1), TCM-199 buforowany HEPESem (20) lub TYH wzbogacony o BSA (29). Inni autorzy stosują do płukania bardziej złożone pożywki, w skład których prócz surowic lub albumin surowiczych (źródło białka w pożywkach) wchodzą również hormony, antybiotyki i substancje grzybobójcze: Otoi i wsp. ...
... Wybiera się w pełni ukształtowane pęcherzyki drugorzędowe (preantralne/APAN) i trzeciorzędowe (antralne/AN) oraz wczesne pęcherzyki trzeciorzędowe (antralne/EAN) w celu wykorzystania ich struktury do IVM (2, 3). Wybór ten podyktowany jest faktem, iż (jak dowiodły badania) zdecydowana ich większość zawiera oocyty w metafazie I podziału mejotycznego o cytoplazmie posiadającej wyraźne zaciemnienia wynikające z obecności lipidów (co jest jednym z kryteriów wyboru oocytów do IVM) (2,3,8,14,29). Ponadto, jak dowiedli Songsasen i Wildt (26), oocyty pochodzące z większych pęcherzyków jajnikowych wykazują większą zdolność do wznowienia mejozy (80% oocytów o średnicy > 2 mm wznowiło mejozę, pozyskanie oocytów z mniejszych pęcherzyków skutkowało albo bardzo niewielką liczbą wznowień mejozy, albo ich brakiem) (26). ...
As a consequence of the untypical characteristics associated with the reproductive physiology of dogs the research on the IVP in this species has not been effective. The whole IVP procedure is preceded by a stage of oocytes collection, the quantity and quality of which may in the future affect the number and ability of the ' development of embryos. In the presented paper methodologies used in the collection, evaluation and selection of oocytes in dogs are summarised.
... Dans ces espèces, un traitement de superovulation est en général administré à la femelle donneuse pour augmenter le nombre d'embryons à collecter. Cependant, la chienne ne répond pas ou mal aux traitements hormonaux de superovulation classiquement utilisés (eCG/hCG ; Archbald et al. 1990 ;Yamada et al. 1992). D'autre part, jusqu'à 9 jours après la fécondation, la collecte des embryons s'effectue par rinçage des oviductes et donc nécessairement par voie chirurgicale, donc invasive. ...
... La collecte des ovocytes peut s'effectuer par ponction de follicules préovulatoires (Yamada et al. 1992 ) (figure 3). Elle peut également se réaliser par section, à l'aide d'une lame de rasoir, des ovaires prélevés par ovariectomie (Reynaud et al. 2004) : cette modalité est celle qui est la plus couramment utilisée pour les études fondamentales menées sur l'ovocyte canin et sa maturation in vitro. ...
... La plupart (60 %) n'ont même pas repris leur méiose (Saint-Dizier et al. 2001 ;Luvoni et al. 2005). Avec des ovocytes issus de follicules préovulatoires, le taux de maturation final est de 32 % (Yamada et al. 1992). ...
La biologie de l’ovocyte et de l’embryon canins, qui présentent des particularités spécifiques, est très mal connue. La chienne se distingue principalement par les modalités de reprise de la méiose ovocytaire : celle-ci n’a pas lieu au moment de l’ovulation, comme chez les autres femelles mammifères, mais dans l’oviducte 48 à 60 heures après l’ovulation. Les facteurs responsables de ce retard ne sont pas connus. Ceci explique sans doute pourquoi les taux de maturation in vitro obtenus à l’heure actuelle sont faibles (10 à 30 %). La collecte des ovocytes est aussi un facteur limitant majeur, en l’absence de protocole efficace d’induction des cycles et de superovulation. Les rendements de fécondation in vitro sont également faibles (10 %), avec un taux élevé de polyspermie. À l’heure actuelle, aucun chiot n’est encore né à partir d’un embryon produit in vitro. Quant aux embryons produits in vivo, leur collecte est difficile pour des raisons anatomiques et le rendement est limité par l’impossibilité d’induire des superovulations ; par ailleurs, leur transfert chez des femelles receveuses se heurte aux difficultés de synchronisation des ovulations entre la femelle donneuse et les receveuses, et la littérature ne décrit que six essais, qui ont abouti à la naissance de 45 chiots. Avec un très faible recours à la culture in vitro, quatre naissances de chiots ont été obtenues par clonage de cellules somatiques. Les biotechnologies de la reproduction sont donc largement en retard dans l’espèce canine, qui souffre d’un manque de travaux fondamentaux visant à mieux comprendre ses mécanismes physiologiques spécifiques. Ce déficit est d’autant plus dommageable que le chien prend une place croissante et pertinente en tant que modèle biomédical.
... Capacitated spermatozoa display a hyperactivated pattern of motility, which is characterized mainly by an accentuated curvilinear line velocity and lateral head displacement. Capacitation time varies between species, and has been found to be 3 to 7 hours for dog spermatozoa when studied in different culture media in vitro (Mahi and Yanagimachi, 1976; 1978; Yamada et al., 1992; Kawakami et al., 1993; Guèrin et al., 1999). Rota et al., (1999 found that the preservation of dog spermatozoa by extending and chilling, or freezing and thawing significantly shortened the time for capacitation-like changes from 4 hours in fresh semen to 2 hours in the preserved samples. ...
... Unlike most other mammals the dog ovulates primary oocytes, that are at the beginning of the first meiotic division, at metaphase I (MI) and the germinal vesicle break down (GVBD) takes place shortly thereafter. In vivo, canine oocytes mature in the oviducts and there is a multilayered and tight cumulus mass around the oocyte, which is seen to expand as the oocyte matures, a process that takes 2 to 5 days to complete (Holst & Phemister, 1971; Mahi & Yanagimachi, 1976; Yamada et al., 1992;).The oviductal transit takes 5-10 days (Andersen and Simpson, 1973;). Fertilization occurs during this passage, in the distal part of the oviduct. ...
... Various culture media have been used: TCM 199, Hams F-10 with BSA or homologous serum, and Krebs Ringer Lactate. There are conflicting results concerning the beneficial effects of the addition of hormones such as FSH, LH, oestradiol or progesterone to the culture media on the maturation rate (Yamada et al., 1992; Nickson et al., 1993; Hewitt and England, 1999). It has been suggested that the granulosa cells may contain an FSH-dependent pathway that is capable of controlling oocyte maturation (Kalab et al., 1997). ...
The dog has been used in medical research as a model for humans, but the lack of appreciation of the differences especially in reproductive pattern and hormonal effects and sensitivity has led to some classic misconceptions for instance regarding the tumorigenic effects of progestagens on mammary glands. In contrast, the dog has proved to be a very useful model in studies on human prostatic functions. The domestic dog is also more and more used as a model in research aiming at the preservation of the many species of wild canids that are threatened by extinction; projects popularly referred to as "The Frozen Zoo". During the last 10-15 years our knowledge of canine reproduction has made major progress. This chapter aims at summarising the basic reproductive physiology of the dog, including the latest discoveries within this field, and also to give an update on the applications of new reproductive technology in this species. Reproductive endocrinology The reproductive events, both in the male and the female dog, are orchestrated from the hypothalamus which in response to some as yet partly unknown stimuli produces and releases the gonadotrophin-releasing hormone (GnRH), which, in turn, influences the pituitary gland to secrete follicle stimulating hormone (FSH) and luteinizing hormone (LH). These gonadotrophic hormones induce ovarian follicular development and ovulation in the bitch and testicular development, androgen production and spermatogenesis in the male. The hypothalamic-pituitary-gonadal axis is regulated via intricate feedback mechanisms (Fig 1a, b) whereby the gonadal hormones having reached a certain concentration via negative feedback downregulate further release of GnRH, and thus FSH and LH. Reproduction in the Male The reproductive organs of the male dog consist of the testicles with epididymides and the vas deferens, the prostate gland, the urethra and the penis. The testicle contains the seminiferous tubules, producing spermatozoa, and the interstitium with Leydig cells which produce steroids, particularly testosterone, in the sexually mature individual. The epididymis consists of a single long duct in which the spermatozoa during passage undergo maturational changes and obtain the capacity for motility. The distal part of the epididymis, the cauda, is the storage site for the matured spermatozoa. Prostatic fluid constitutes the major portion of the ejaculate, and contains several enzymes, cholesterol and lactate. The penis consists of a pelvic part, and
... In the literature the biology of the canine oocyte cell was usually compared with that of other mammalian females but, in contrast to oocytes of most mammals, the canine oocyte is at the germinal vesicle stage at ovulation [3,4,5]. The knowledge of the cumulus-oocyte complex characteristics plays an important role for in vitro fertilization and not only. ...
... All the oocytes investigated from the canine ovaries were at the germinal vesicle stage, so immature oocytes. According to the literature [3,4,5], in contrast to oocytes of most mammals, the canine oocyte is at the germinal vesicle stage at ovulation. In Fig. 3 we report an image with an immature canine oocytes surrounded by compact cumulus cells. ...
In this paper we report the results concerning the refractive index of canine oocytes. To determine the refractive index of canine oocytes we have used two theoretical models based on matrix optics. For this we considered the oocyte as a microlens and having a spherical shape. In our work we performed and a microscopic study using an inverted microscope Nikon Eclipse Ti-U in order to determine the oocyte dimensions and to obtain the information regarding the maturity of canine oocytes.
... Several studies have demonstrated the link between the inclusion of L-Carnitine during IVM and the reduction in intracellular ROS and the increase in the antioxidant enzymes within the oocyte of many species, including mice [37], cattle [40], pigs [21,31], and sheep [34]. The nuclear maturation rates reported here in 0.3 and 0.6 mg/mL LC supplemented groups (35.4% to 41.4%) were higher than those reported previously for dog oocytes cultured in vitro for 48 h (11.9% to 18.6% [27]; 2.2% to 13.3% [41]; 8.4% to 14.7% [7]; 25.6% [9]; 27.3% [42]; and 11.7% to 17.7% for oocytes collected from small (<1 mm) and medium (1-2 mm) follicles, respectively [15]). However, in the study of Songsasen et al. [15], the collection of oocytes from large (>2 mm) follicles resulted in comparable maturation rates (37.7%) to our 0.3 and 0.6 mg/mL LC groups. ...
... Attempts at IVF of IVM dog oocytes previously resulted in low embryo development rates and no live births [8,9,12,24,26,27,47]. For example, only 2% of inseminated dog IVM oocytes developed to eight-cell embryos at 72 h post-IVF [42]. Otoi et al. [8] reported that only one canine oocyte developed to the blastocyst stage following IVM/IVF and IVC. ...
This study aimed to investigate the effect of L-carnitine (LC) supplementation during in vitro maturation (IVM) of canine oocytes on nuclear maturation, fertilization status, and preimplantation development. Cumulus–oocyte complexes (COCs) collected from the ovaries of ovariohysterectomized female dogs were matured in vitro for 72 h in a TCM-199 medium supplemented with (0.1, 0.3, 0.6, 1.0, or 2.0 mg/mL) or without (0.0 mg/mL) LC. Matured oocytes were fertilized in vitro with frozen–thawed spermatozoa, and zygotes were cultured in a SOF medium for 7 days. IVM rates were higher (p ≤ 0.05) in 0.3 and 0.6 mg/mL LC supplemented groups than in the control (0.0 mg/mL LC) and other LC groups. Fertilization (18 h postinsemination (pi)) and cleavage (2–16-cell stage at day 3 pi) rates were higher (p ≤ 0.05) in the 0.6 mg/mL LC group than in the control and 0.1, 1.0, and 2 mg/mL LC supplemented groups. Interestingly, 4.5% of fertilized oocytes developed to morula (day 5 pi) in the 0.6 mg/mL LC group, which was higher (p ≤ 0.05) than those developed in the 0.3 mg/mL group (1.0%). No cleaved embryos developed to morula in other groups. In conclusion, LC supplementation at 0.6 mg/mL during IVM of canine oocytes improved their maturation, fertilization, and preimplantation embryo development rates following IVF and in vitro culture (IVC).
... 43 Thus, using oocytes from large follicles makes it possible to increase the proportion of fertilizable oocytes after in vitro maturation. 44 Yet, rates of success remain low, which may be related to cytoplasm quality (e.g. number and organization of organelles such as mitochondria and cortical granule) in those in vitro-matured oocytes reaching the metaphase II stage, which remain largely below those obtained with oocytes ovulated in vivo. ...
... 66,67 Several teams have described in vitro fertilization and occasionally obtained embryos and even puppies. 27 However, bringing together dog sperm and oocytes in vitro does not routinely lead to successful fertilization, because sperm can enter immature oocytes, 64,68 typically ending in a high rate of polyspermia 44,69 and sperm pronucleus abnormalities. 37,65 However, oocytes collected at follicular phase stage have been used successfully to obtain embryos with as many as 30% undergoing cleavage after fertilization using oocytes from follicles > 2 mm. ...
Although the dog and cat both belong to the order Carnivorous, their reproductive physiology is quite different. The dog is a nonseasonal, monoestrus species with spontaneous ovulation only twice a year and with an atypical, postovulatory oocyte maturation. Furthermore, the application of reproductive in vitro biotechnologies such as in vitro maturation (IVM), in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) or cloning remains quite a challenge in dogs. By contrast, the cat is a polyestrus seasonal species, with ovulation induced several times a year and typical preovulatory oocyte maturation. As a result, manipulation of ovarian physiology is feasible and in vitro reproductive biotechnologies are as efficacious as in cattle. The first part of this review describes the main facts associated with folliculogenesis in the dog and cat (initiation of growth of primordial follicles, appearance of the zona pellucida, formation of antrum, nuclear and cytoplasmic maturation of oocyte, expression of receptors to gonadotrophins, and expression of steroidogenic enzymes). Second part focuses on oocyte maturation, fertilization, and in vivo early embryo development in both species. Last part discusses in vitro reproductive biotechnologies (IVM, IVF, and IVD), embryo transfer, and more recent biotechnologies (cloning, in vitro folliculogenesis, and vitrification of oocytes and follicles). Innovations in the dog are still limited by various characteristics (long ovarian cycle, difficult in vitro oocyte maturation) whereas in the cat, many in vitro techniques are applicable that may also be extended to wild feline species. Introduction Although the dog and cat belong to same order (Carnivorous), their reproductive physiology is quite different. Dog is nonseasonal monoestrus, with spontaneous ovulation only twice a year. Thus, manipulation of ovarian physiology (e.g. to reduce interval between ovulations, to induce ovulation or superovulation, or to produce embryos) is generally difficult. Furthermore, application of reproductive in vitro biotechnologies (in vitro maturation [IVM], in vitro fertilization [IVF], intracytoplasmic sperm injection [ICSI], and cloning) remains quite a challenge in the dog and only a few research teams have mastered them. By contrast, the cat is seasonally polyestrus with ovulation induced several times a year. As a result, manipulation of ovarian physiology (e.g. stimulation or induction of ovulation) is feasible and in vitro reproductive biotechnologies are as efficient as in cattle. Our purpose is to review available information on: 1) main events occurring during in vitro follicle and oocyte growth, in vivo fertilization, and early embryo development; 2) in vitro biotechnologies and embryo transfer; and 3) other biotechnologies.
... 43 Thus, using oocytes from large follicles makes it possible to increase the proportion of fertilizable oocytes after in vitro maturation. 44 Yet, rates of success remain low, which may be related to cytoplasm quality (e.g. number and organization of organelles such as mitochondria and cortical granule) in those in vitro-matured oocytes reaching the metaphase II stage, which remain largely below those obtained with oocytes ovulated in vivo. ...
... 66,67 Several teams have described in vitro fertilization and occasionally obtained embryos and even puppies. 27 However, bringing together dog sperm and oocytes in vitro does not routinely lead to successful fertilization, because sperm can enter immature oocytes, 64,68 typically ending in a high rate of polyspermia 44,69 and sperm pronucleus abnormalities. 37,65 However, oocytes collected at follicular phase stage have been used successfully to obtain embryos with as many as 30% undergoing cleavage after fertilization using oocytes from follicles > 2 mm. ...
... Several studies have demonstrated the link between the inclusion of L-carnitine during IVM and the reduction in intracellular ROS and the increase in the antioxidant enzymes within the oocyte of many species, including mice [37], cattle [40], pigs [21,31], and sheep [34]. The nuclear maturation rates reported here in 0.3 and 0.6 mg/mL LC supplemented groups (35.4% to 41.4%) were higher than those reported previously for dog oocytes cultured in vitro for 48 h (11.9% to 18.6% [27]; 2.2% to 13.3% [41]; 8.4% to 14.7% [7]; 25.6% [9]; 27.3% [42]; and 11.7% to 17.7% for oocytes collected from small (<1 mm) and medium (1-2 mm) follicles, respectively [15]). However, in the study of Songsasen et al. [15], the collection of oocytes from large (>2 mm) follicles resulted in comparable maturation rates (37.7%) to our 0.3 and 0.6 mg/mL LC groups. ...
... Attempts at IVF of IVM dog oocytes previously resulted in low embryo development rates and no live births [8,9,12,24,26,27,47]. For example, only 2% of inseminated dog IVM oocytes developed to eight-cell embryos at 72 h post-IVF [42]. Otoi et al. [8] reported that only one canine oocyte developed to the blastocyst stage following IVM/IVF and IVC. ...
In vitro embryo production (IVP) in the domestic bitch is important for conservation of endangered canids. Compared with various domestic animals, the development of assisted reproductive technologies (ART) in the dog has lagged behind, mainly due to the low percentage of oocytes that can reach metaphase II (MII) stage after in vitro maturation (IVM). Beneficial effects of l-carnitine (LC) on embryonic development in culture have been reported in many mammalian species; however, no studies have been conducted in dogs. The aim of the present study was to investigate the effect of LC supplementation during IVM of canine oocytes on nuclear maturation, fertilization status, and pre-implantation development following IVM/IVF. Cumulus-oocyte complexes (COC) were collected by slicing ovaries obtained from dogs (n = 20, 1 to 6 years of age) after ovariohysterectomy. The COC were subjected to IVM for 72 h in a medium (TCM-199) supplemented with LC at different concentrations (0.1, 0.3, 0.6, 1.0, or 2.0 mg mL−1) or without LC supplements (0 mg mL−1; control). Matured oocytes were fertilized in vitro with frozen–thawed spermatozoa, and presumptive zygotes were cultured in SOF medium for 7 days. Frequencies of nuclear maturation (72 h post-IVM), fertilization rates (18 h post-insemination), and embryo development (Days 2 to 5 post-insemination) were evaluated. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparisons test. Supplementation of IVM medium with 0.3 or 0.6 mg mL−1 LC significantly improved (P ≤ 0.05) maturation (35.4% and 41.4%) and fertilization (21.3% and 25.8%) rates compared with the controls and with other LC-supplemented groups; values ranged from 20.1% to 25.0% for maturation and from 12.1% to 14.6% for fertilization. Cleavage (2- to 16-cell stages) was significantly higher (P ≤ 0.05) in the 0.6 mg mL−1 LC supplemented group than the 0.3 mg mL−1 supplemented group (16.3% v. 13.3%). These values were significantly higher (P ≤ 0.05) than those in other groups. Interestingly, 4.5% of IVM/IVF oocytes were developed to morula in 0.6 mg mL−1 LC supplemented group which was significantly higher (P ≤ 0.05) than those developed in the 0.3 mg mL−1 supplemented group (1.0%). No embryos developed beyond the 2- to 16-cell stage in the rest of the groups. In conclusion, l-carnitine supplementation during IVM is particularly efficient in improving nuclear maturation and pre-implantation embryo development of canine oocytes after IVF. These outcomes are important for the improvement of IVM conditions that can advance the efficiency of ART in dogs.
... in the cytoplasm are limited and contradictory. Van der Stricht (1923) reported very little modification of the penetrated sperm head until the expulsion of the second polar body. Mahi and Yanagimachi (1976) observed swelling of the sperm nuclei in the cytoplasm of oocytes at all maturation stages with no difference in the degree of decondensation. Yamada et al. (1992) observed an enlarged sperm head in oocytes arrested at the germinal vesicle stage, but a male pronucleus was observed only in oocytes that had a female pronucleus. Renton et al. (1991) observed two structures in a one-cell embryo recovered 5 days after assumed ovulation; these two structures were determined to be pronuclei, but no furth ...
... Fertilization at the germinal vesicle stage implies the entry of a highly compacted haploid male nucleus into the female gamete at a stage when the chromatin is partially condensed. The finding that the sperm head is moderately enlarged at the germinal vesicle stage and forms a male pronucleus when female chromatin has reached the pronuclear stage supports the observations of Yamada et al. (1992 Yamada et al. ( , 1993). Nevertheless, unlike Van der Stricht (1923) and Mahi and Yanagimachi (1976), parallel modification of male and female chromatin were observed during the oocyte maturation process. ...
... While some researchers report no association (ROBERTS & RODRIGUES, 2003;APPARÍCIO et al., 2011), others show that bitch reproductive status influences the ability of oocyte development (OTOI et al., 2001;KIM et al. 2004). Such that, oocytes obtained during the follicular phase (proestrus and estrus) show better maturation indexes compared to oocytes retrieved from animals at other reproductive stages (YAMADA et al. 1993;OTOI et al. 2001;KIM et al., 2004;SONGSASEN & Wildt, 2007). ...
... Due to the difficulties incurred in the IVM phase, IVF cleavage and blastocyst rates in dogs are still very low. Yamada et al. (1993) reported that only 2% of inseminated oocytes reached the eight-cell stage. Otoi et al. (2000) obtained one blastocyst out of 217 inseminated oocytes and England et al. (2001) obtained a single IVF pregnancy in this species. ...
The development of assisted reproductive techniques (ART) in dogs and cats is an area of increasing interest because ART can be potentially applied to non-domestic carnivores threatened with extinction. Much progress has been achieved in ART of the feline species. However, advances on assisted reproduction in dogs are hampered by its low efficiency. A major obstacle lies on the particular characteristics of canine gamete physiology, for instance, the highly complex requirements for oocyte maturation and development, which makes difficult to apply protocols/techniques that have been successful in other species. This review aims to depict the advances made on assisted reproductive techniques in dogs and cats, focusing on those features that may be relevant for applying these techniques to wild endangered carnivores. KEY-WORDS: Bitch. Cat. In vitro embryo production.
... Oocyte maturation and fertilization in these species are unique among mammals. Ovulation occurs anytime between Days 2 and 7 after the initiation of estrous behavior although it most frequently happens between Days 1 and 3 (Yamada et al., 1992). Normally, oocytes are ovulated in the germinal vesicle stage, and the first meiotic division is not completed until at least 48 hours after ovulation into the uterine tubes (Van der Stricht, 1923;Tsutsui and Shimizu, 1975). ...
... In canids, in vitro maturation has limited success with maturation rates varying from 0 to 58% (Farstad, 2000b). The most common canine IVM culture media is TCM 199 (Hewitt and England, 1997a;Hewitt et al., 1998;Otoi et al., 2000a;b;Rodrigues and Rodrigues, 2002a;b;2003;Songsasen et al., 2002;Luvoni et al., 2003;Willingham-Rocky et al., 2003) although modified Krebs ringer bicarbonate (Yamada et al., 1992(Yamada et al., , 1993 and synthetic oviduct fluid (Hewitt and England, 1999a;b;Bolamba et al., 2002) could also be used. ...
The present study evaluated nuclear maturation after in vitro culture of oocytes originated from estrous and anestrous bitches. Two culture media were used, TCM 199 (tissue culture medium 199) and synthetic oviduct fluid (SOF). The females were divided in two groups. Group 1 consisted of 11 anestrous bitches, and Group 2 consisted of 6 estrous bitches. In each group, oocytes were harvested from sliced ovaries, and one- half of them were immediately stained with Hoechst 33342. The oocytes were classified as in the GV (germinal vesicle) phase, GVBD (germinal vesicle breakdown) phase, MI (metaphase I), or as D/NI (degenerated/non identifiable). The other half of the oocytes were matured in SOF or TCM media for 24 hours, stained, and evaluated as previously described. There were 511 and 373 Grade I oocytes recovered in Groups 1 and 2, respectively. The mean number of oocytes obtained per anestrous bitch was 46.5 ± 25.2. For females in Group 2 (estrous), 62.2 ± 8.2 oocytes were obtained per bitch. Oocytes from estrous and anestrous bitches submitted to in vitro maturation had similar rates of GVBD (21.7%, TCM 199 and 23.6%, SOF - anestrous; 20%, TCM 199 and 23%, SOF - estrous). However, the MI rate was higher in the estrous group (9.8%, TCM 199 and 9.7%, SOF - anestrous; 25%, TCM 199 and 58%, SOF - estrous). The protocol that utilized estrous donor's oocytes and in vitro maturation in SOF was superior to the other protocol tested, demonstrating that the association between estrous versus anestorus and maturation medium is beneficial for oocyte in vitro maturation (58% MI). It was demonstrated that 24 hours of in vitro maturation was insufficient for obtaining Metaphase II nuclear configurations in oocytes with all of the protocols tested.
... In several mammalian species, in vitro fertilization (IVF) of matured oocytes is well established and used routinely7475767778. In dogs however, in vitro maturation (IVM) and fertilization (IVF) of oocytes is difficult to achieve (for review see [79,80]) and, despite the numerous attempts to improve the results (for review see [17,80]), the percentages of canine oocytes which are fertilised in vitro are generally very low [6,41,71,81828384. Additionally, further embryonic development did not occur except in studies performed by Yamada et al. [83] and Rodrigues et al. [41] in which, respectively 2 and 0.75% of the fertilised oocytes reached the eight-cell stage. ...
... In dogs however, in vitro maturation (IVM) and fertilization (IVF) of oocytes is difficult to achieve (for review see [79,80]) and, despite the numerous attempts to improve the results (for review see [17,80]), the percentages of canine oocytes which are fertilised in vitro are generally very low [6,41,71,81828384. Additionally, further embryonic development did not occur except in studies performed by Yamada et al. [83] and Rodrigues et al. [41] in which, respectively 2 and 0.75% of the fertilised oocytes reached the eight-cell stage. Recently, one study produced a single blastocyst [85] and England et al. [86] reported a pregnancy following IVF in the bitch with subsequent embryo resorption. ...
... This glycoprotein is a major nonserum protein present in the oviductal lumen at the time of ovulation, fertilization and early embryonic development [9]. Researchers reported that bitch preovulatory oocytes treated with gonadotrophins achieved the highest maturation rates (32% MII) and after in vitro fertilization, 30% of embryos reached the 2-8 cell stage in in vitro culture [10]. The ovulated immature dog oocyte completes its nuclear maturation in 48 -72 h after the LH peak in vivo and canine spermatozoa are also able to penetrate the zona pellucida and vitellus in vitro and in vivo , irrespective of the oocyte maturation stage [10]. ...
... Researchers reported that bitch preovulatory oocytes treated with gonadotrophins achieved the highest maturation rates (32% MII) and after in vitro fertilization, 30% of embryos reached the 2-8 cell stage in in vitro culture [10]. The ovulated immature dog oocyte completes its nuclear maturation in 48 -72 h after the LH peak in vivo and canine spermatozoa are also able to penetrate the zona pellucida and vitellus in vitro and in vivo , irrespective of the oocyte maturation stage [10]. It is demonstrated that sperm penetration can occur in immature canine oocytes and that it induces resumption meiosis [11]. ...
There have not been successful and repeatable methods for in vitro embryo production in the dog. Up to date, only one blactocyst has been achieved on in vitro culture. Since reproductive physiology of the dog is different from that of other mammalian species, it seems that a suitable method for in vitro production of canine embryos is still far from being designed and routinely applied, and an effective protocol is needed. Therefore, the aim of the present study was to examine the effects of adding hormones sequentially, for mimicking the dog's in vivo endocrine milieu, on maturation of immature dog oocytes in vitro. At the end of the 96 h IVM period, nuclear maturation rates were evaluated by the aceto-orcein staining method. In comparison relating IVM rates, the sequential hormone addition was more beneficial on IVM rates (MI MII) than the traditional hormone addition and control groups (48.1%, 38.9% and 23.0% respectively; P 0.0001). As a result, hormone addition sequentially may be an effective approach for the IVM of the immature dog oocytes. We suggest that attempts to define the adequate conditions for IVM in the dog should extend towards this new perspective.
... ile COCs from bitches in anestrus have closed GAP junctions, while COCs from bitches at the end of proestrus have 89% of opening in communicating junctions(Luvoni, Luciano, Modina, & Gandolfi, 2001). This finding could justify the low maturation rates of oocytes from bitches in anestrus, even after a culture period of 72 hr, observed in this study.Yamada et al. (1992) demonstrated that oocytes from anestrous bitches have no tendency to resume meiosis in vitro. A similar result was described byLuvoni et al. (2001), indicating that dog COCs isolated from anestrous ovaries are unable to reinitiate meiosis when in culture. Despite these results, our earlier study showed that approximately 19% of the oocy ...
In domestic dogs, oocyte maturation rates are low and the percentage of oocytes that remain in the stage of germinal vesicle (GV) regardless of culture conditions is high. The present study was conducted to characterize the proteome of canine oocyte at the germinal vesicle stage using label-free mass spectrometry. Ovaries were collected from 415 adult domestic dogs and oocytes were divided anestrus and diestrus group. Protein lysates were subjected to quantitative proteomic analysis to identify differentially expressed proteins in different status reproductive. All runs for each sample were performed on an Easy nLC1000 nano-LC chromatograph system directly connected to a quadrupole-type Orbitrap mass spectrometer. For identification of peptides and proteins, raw data of the spectra were loaded into MaxQuant software version 1.5.2.8. Proteomic data were analysed according to gene ontology and a protein-protein interaction network. 312 proteins were identified and grouped according to their biological processes, molecular functions and cellular component. Forty-six differentially expressed proteins among diestrus and control group were associated with at least one GO term in the biological process database. Several proteins involved in the cell cycle, fertilization, regulation of transcription and signalling pathways that are essential for the full development of oocytes and fertilization were expressed. This study identified proteins that were absent, and more or less expressed in different status reproductive. These differentially expressed proteins revealed a framework of molecular reorganization within a GV that renders its competency. This knowledge will enable the identification of target competence biomarkers and thus the establishment of more adequate means of cultivation to improve the M-I and II indexes in this species and also to better understand the physiology of the domestic dog, promoting the development of new reproduction biotechniques.
... This higher value may be attributed to the use of a bitch serum containing progesterone (1.6 ng/ml) and estradiol (31.5 pg/ml) during in vitro culture (IVC). Similar to our results, Yamada et al. (1992) found that 48 h post insemination 14.6% of the oocytes were at the 2-3-cell stage and 72 h post insemination 4.8% of the oocytes were at 5-8-cell stage. ...
The aim of the current study was to investigate the effect of caffeine supplementation during in vitro maturation (IVM) for different maturation times on the developmental potential of canine oocytes recovered from ovariohysterectomized bitches. The recovered cumulus–oocytes complexes were in vitro matured for 72 h. Here, 10 mM caffeine was added to the maturation medium for different incubation times (caffeine from 0–72 h maturation, caffeine for the first 24 h of maturation only, caffeine addition from 24 to 48 h maturation time, caffeine addition from 48 to 72 h maturation or in caffeine-free medium, control group). The matured oocytes were in vitro fertilized using frozen–thawed spermatozoa. The presumptive zygotes were in vitro cultured in synthetic oviductal fluid medium for 5 days. The results showed that both maturation and fertilization rates were significantly higher ( P ˂ 0.05) using caffeine-treated medium for the first 24 h of maturation compared with the control and other two groups of caffeine treatment (from 24 to 48 h and from 48 to 72 h), whereas use of caffeine-treated medium for a 0–72 h incubation time did not affect these rates ( P > 0.05). Interestingly, the matured oocytes in caffeine-supplemented medium for the first 24 h or from 0–72 h showed a significant ( P ˂ 0.05) increase in the total number of cleaved embryos compared with the control group. In conclusion, supplementation of the maturation medium with 10 mM caffeine for the first 24 h of maturation or during the whole maturation time (0–72 h) improved nuclear maturation and subsequent embryo development preimplantation following in vitro fertilization.
... These results show that the time of 72 h reflects the optimum culture time to optimize oocyte maturation. These results corroborate the fact that oocytes in bitches are ovulated still at the immature stage, needing after ovulation from 2 to 5 days to complete maturation (YAMADA et al., 1992). In this context EGF seems to be necessary for competence acquisition since the chances of obtaining oocytes at M-I and M-II stages were higher in the groups supplemented with this growth factor. ...
The aim of this study was to evaluate the influence of epidermal growth factor (EGF) on in vitro maturation of canine oocytes at different times of the process. Ovaries were collected from 55 bitches considered healthy and aseptically isolated, immersed in physiological solution (0.9% NaCl) and transported under refrigeration. Grade 1 cumulus-oocyte complexes (COCs) were selected and divided into two groups: control group (CG) and treatment group (TG). In CG 698 grade I COCs were placed in 4-well plates containing TCM-199 medium supplemented with 25 mM HEPES, 100 IU/mL penicillin, 100 mg/mL streptomycin, 26 mM sodium bicarbonate, 1.5 mM sodium pyruvate, 2.9 mM sodium lactate pentahydrate, 0.6 mM cysteine, 0.03 IU/mL hCG, 0.5 µg/mL FSH, 20 µg/mL estrogen at 38.5oC in a humidified atmosphere of 5% CO2 in times of 24 h, 48 h, and 72 h. In TG 547 COCs received the same maturation medium plus 10 ηg/mL EGF. Logistic regression models (SAS, 2011) were constructed in order to estimate the chances of oocytes being observed at nuclear maturation stages in different culture times (24 h, 48 h, and 72 h). Based on the results found EGF-supplemented medium showed 2.56 times more chances of having an oocyte at metaphase I (M-I) than medium without EGF (p < 0.0001). The results of this study demonstrated that the time of 72 h showed 5.88 times more chances of having an oocyte at metaphase II (M-II) compared to time of 24 h (p = 0.0001) and 7.69 times more chance than time of 48 h (p = 0.0001). The chances of finding an oocyte at M-II were also 9.09 times higher in medium supplemented with EGF than in medium without EGF (p = 0.0001). Thus, these results demonstrated the essential importance of EGF at different moments of oocyte maturation, being a key component for the acquisition of meiotic competence in bitches, increasing the M-I and M-II rates. © 2017, Universidade de Sao Paulo. Faculdade de Medicina Veterinaria e Zootecnia. All rights reserved.
... Além disso, esses oócitos são ovulados ainda imaturos, no início da primeira divisão meiótica (M-I) e logo após há a quebra da vesícula germinativa. Assim, após a ovulação, os oócitos requerem de 2 a 5 dias para completar a maturação (YAMADA et al., 1992). Desse modo, a maturação completa e a fecundação ocorrem dentro do oviduto e a aquisição da competência meiótica ocorre em ambiente intra e extrafolicular (LUVONI et al., 2005). ...
A cadela apresenta particularidades reprodutivas que a diferencia de outras espécies. Diversos experimentos têm sido realizados visando estabelecer protocolos eficientes para a maturação, entretanto os resultados mostram-se insatisfatórios. Nesse aspecto, a fase reprodutiva da fêmea doadora deve ser considerada, já que pode ser um fator de variabilidade dos achados ate então presenciados nessa espécie. O objetivo deste estudo foi avaliar a influencia das fases do ciclo estral (anestro e diestro) na maturação in vitro (MIV) de cadelas. Os ovários foram transportados em solução de cloreto de sódio 0,9% e seccionados em solução salina fosfato tamponado (PBS) e os complexos cumulus-oócito (COCs) selecionados em meio de cultura de tecidos (TCM) 199 suplementado com HEPES. Foram obtidos 469 oócitos grau 1 de cadelas em anestro e diestro. Esses oócitos selecionados foram transferidos para o meio de maturação por um período de 72 horas, sendo posteriormente submetidos a solução de hialuronidase e corados com HOESCHT 33342 para avaliação da configuração nuclear. A comparação das fases de anestro e diestro não revelou diferença (p > 0,05) entre os estágios de maturação nuclear. Dessa maneira, a fase do ciclo estral não influenciou na maturação in vitro de oócitos caninos, incrementando os índices de M-II nesta espécie.
... Sperm capacitation has long been recognized as a timedependent, species-dependent phenomenon [23]. Similar to human [24] sperm, domestic cat sperm appear to require a shorter time (2-2.5 h) to capacitate than ejaculated sperm of pigs (4-5 h) [25], bulls (4 h) [26], or dogs (4-5 h) [27]. Additionally, the conditions for in vitro capacitation of domestic cat sperm are less complex than for other species that require exposure to a hypertonic medium [281, to heparin [261, or to a high calcium concentration [25]. ...
The efficiency of sperm capacitation and of the acrosome reaction was studied in the teratospermic domestic cat to evaluate further the etiology of compromised zona pellucida penetration and oocyte fertilization. Specific objectives were to compare normospermic and teratospermic cat ejaculates for 1) the kinetics and timing of sperm capacitation in vitro as determined by an ionophore-induced acrosome reaction; 2) the incidence of spontaneous acrosomal loss; 3) the ability of capacitated, swim-up processed sperm to acrosome-react in response to chemical (calcium ionophore) or physiological (solubilized zonae pellucidae) inducers; and 4) differences in acrosomal ultrastructure by use of transmission electron microscopy (TEM). Acrosomal status was determined with the fluorescent probe Arachis hypogaea (peanut) agglutinin. The timing of in vitro capacitation differed (p 0.05) between cat groups (range, 7.6-17.8%). In swim-up separated sperm from normospermic cats, ionophore A23187 was a more potent inducer (p 0.05). Sperm motility patterns over time indicated that differences in acrosomal status were not influenced by cell death. The frequency of abnormal acrosomes detected by TEM was higher (p 0.05) the proportion of sperm cells with malformed acrosomes (swim-up, 33.5 3.5%; washed, 26.6 4.6%). These results indicate that sperm from teratospermic cats exhibit a high incidence of malformed acrosomes detectable only at the ultrastructural level. Nevertheless, acrosomal dysfunction is not related exclusively to structural defects because > 40.0% of swim-up separated sperm with structurally normal acrosomes still are incapable of completing the acrosome reaction. This suggests that compromised capacitation and acrosomal dysfunction may be responsible for low fertilization success in the teratospermic domestic cat.
... Mahi and Yanagimachi [6] found that canine sperm required capacitation before they were able to penetrate the ZP. Yamada et al. [36] found that canine sperm preincubated for 4 h can begin to penetrate the ZP within 1 h after insemination. Under the capacitation conditions used in these experiments, we have observed spontaneous acrosome reactions in canine spermatozoa after 4 h of incubation, with an additional significant increase after 7 h of incubation [7]. ...
In this study the induction of the acrosome reaction of canine sperm by homologous zona pellucida (ZP) was examined. Twelve semen samples obtained from 6 normal beagle dogs were evaluated after sperm incubation in vitro with canine capacitation medium (CCM). Washed sperm were preincubated at 37 degrees C in 5% CO2 in air for 4 and 7 h prior to experimental treatment. Sperm were co-incubated for 1 min with intact oocytes collected from canine ovaries. Half of the oocytes were then fixed, and the bound sperm were assessed for acrosome reactions through use of a polyclonal antisperm antiserum and indirect immunofluorescence. The remaining oocytes were incubated in sperm-free medium for an additional 1-h period, and the acrosomal status of sperm bound to the ZP was evaluated similarly. The percentage of acrosome-reacted sperm on the ZP increased significantly during the 1-h incubation period. In other experiments, capacitated canine sperm were incubated with heat-solubilized ZP for 1 h and their acrosomal status was determined using fluoresceinated Pisum sativum lectin. The percentages of acrosome-reacted sperm increased significantly in ZP solution compared with controls. These data demonstrate that intact and solubilized canine ZP are capable of inducing acrosome reactions of canine sperm.
... Ivanova et al. (1999) In vitro fertilization (IVF) is another method of evaluating the fertilizing ability of spermatozoa in many mammalian species (Blash et al., 2000;Commizoli et al., 2001;Graff et al., 1996;Nagai et al., 1988;Rath and Niemann, 1997;Songsasen et al., , 1998. Due to the unique physiology of the canine female, IVF has not been successful in canids (Farstad, 2000b), and very few canine embryos have been produced by IVF (Otoi et al., 2000;Yamada et al., 1992Yamada et al., , 1993. However, in a recent study, England et al. (2001) reported a pregnancy following in vitro fertilization of canine oocytes recovered from ovaries at standard ovariohysterectomies. ...
... Yanagimachi (1976, 1978) were the first to report in vitro maturation and sperm penetration of dog oocytes but their success was limited. Yamada et al. (1992) attempted to superovulate dogs before oocyte collection to determine whether maturation and fertilization rates could be improved. The rate of fertilization of the ova was 32% by 72 hours post-insemination, but of the 148 inseminated ova, only 2 developed to the 8-cell stage. ...
... ICSI has equally been poor and has not overcome this dual problem of polyspermy and low fertilization ability (Fulton et al., 1998 ). Although we observed decent in vitro development of in vivo fertilized and flushed canine morulae to the blastocyst stages in SOF medium cultured under 5% oxygen tensions (Wilcox et al., 2009), a 4-to 8-cell block commonly occurs that contributes to their exceptionally poor in vitro development (Yamada et al., 1992; Otoi et al., 2000; Hori and Tsutsui, 2003; Hatoya et al., 2006a). ...
... In pursuit of improving culture conditions of canine oocytes, several attempts have been made during the past decades [1][2][3][4][5][6][7][8]. Maturation rates of canine oocytes are considerably low, and the embryonic development is impaired compared to bovine and ovine species [9]. ...
Phosphodiesterase (PDE) inhibitors have been utilized for in vitro maturation (IVM) of oocytes to manipulate the meiotic resumption and progression. Premature chromatin condensation and DNA replication of the oocytes, immediately after the decrease in the cAMP level, are the difficulties in canine IVM. Caffeine, a nonselective competitive PDE inhibitor, due to its structural similarity to adenosine molecule maintains the cAMP level by occupying PDE enzymes such as PDE-3A inside the oocyte and PDE-4 and PDE-5 in the cumulus cells. In this study, the effects of 12-hour caffeine pretreatment in a biphasic IVM protocol were assessed on maturation rates of canine oocytes. Sixty hours of culture after a 12-hour of 10 mM caffeine pretreatment resulted in 16.9% ± 2.4 of the oocytes reaching metaphase II stage (MII) and 25.9% ± 5.2 degeneration rate compared with the control group with 2.2% ± 2.2 MII and 37.6% ± 4.3 degeneration rates (P < 0.05). Caffeine pretreatment induced higher mitogen-activated protein kinases (MAPK1 and MAPK3) phosphorylation and maturation-promoting factor activity at 12 hours and activated MAPK1 and maturation-promoting factor at 48 hours after culture in cumulus-oocyte complexes (COCs) compared with the control group (P < 0.05). Fresh canine COCs were also analyzed before IVM using brilliant cresyl blue (BCB) staining. Oocytes showed difference in meiotic resumption (MI-MII) (BCB+ = 16.11% ± 5.5, BCB- = 9.86% ± 5.0; P < 0.05) after 60 hours of culture following 12-hour caffeine pretreatment. The BCB+ canine oocytes had higher MII rate than the BCB- group under caffeine pretreatment (10.2% ± 2.9 vs. 1.1% ± 1.1, respectively; P < 0.05). Results indicated that 12-hour caffeine pretreatment of canine COCs improves the MII maturation rates at 72 hours and BCB+ oocytes have higher competency in vitro for nuclear maturation.
... ICSI has equally been poor and has not overcome this dual problem of polyspermy and low fertilization ability ( Fulton et al., 1998). Although we observed decent in vitro development of in vivo fertilized and flushed canine morulae to the blastocyst stages in SOF medium cultured under 5% oxygen tensions ( Wilcox et al., 2009), a 4-to 8-cell block commonly occurs that contributes to their exceptionally poor in vitro development (Yamada et al., 1992;Otoi et al., 2000;Hori and Tsutsui, 2003;Hatoya et al., 2006a). ...
... Nevertheless, the results of nuclear maturation remain low with a percentage of oocytes reaching the Metaphase II stage in the range of 0% to 31.9% (Yamada et al., 1993;Bolamba et al., 1998;Hewitt and England, 1999;Fujii et al., 2000;Saint-Dizier et al., 2001;Bogliolo et al., 2002;Songsasen et al., 2002;Luvoni et al., 2003;Rodrigues and Rodrigues, 2003b;Kim et al., 2005). At the present time, few researchers have had success with in vitro development of canine embryos (Yamada et al., 1992;Otoi et al., 2000b;England et al., 2001;Rodrigues et al., 2004). ...
The main limitation in producing in vitro dog embryos succesfully is the low oocyte maturation rate to the Metaphase II stage. The objective of this experiment was to compare the rates of nuclear maturation of dog oocytes cultured in Tissue Culture Medium 199 (TCM 199) supplemented with polyvinyl-pirrolidone (PVP) with oocytes cultured in TCM 199 with estrous cow serum (ECS) or TCM 199 with ECS and hormones. Ovaries were collected from 21 healthy bitches by ovariohysterectomy. Females were at various stages of the estrous cycle at the moment of ovary retrieval. The oocytes were selected and classified subjectively according to their morphology and size and matured for 48 hours at 37 oC in an atmosphere of 5% CO 2 in air . Oocytes were randomly allocated to one of three treatment groups: (A) TCM 199 supplemented with 4 mg/ml PVP, (B) TCM 199 with 10% ECS, or (C) TCM 199 supplemented with 10% ECS + 20 μg/ml estradiol + 0.5 µg/ml FSH + 0.03 IU/ml hCG (control). All maturation media contained a final concentration of 1 µg/ml of human somatotropin (hST). There were no significant differences in Metaphase II rates among treatments A (4.7%, 8/170), B (3.52%, 6/183), and C (4.70%, 8/172). It was concluded that in vitro nuclear maturation of domestic dog oocytes can be achieved using TCM 199 supplemented with PVP, and this yields results similar to those obtained using media containing serum, gonadotropins, or estradiol.
... In vitro maturation (IVM) systems are well recognized in many mammalian species including pigs and bovine [1][2][3][4]. However, only 20% of immature canine oocytes are able in vitro to resume meiosis and reach the MII stage [5][6][7][8][9]. The poor in vitro development of oocytes and specific features of the estrous cycle of bitches seems to be the main reason for low IVM efficiency [10,11]. ...
The role of progesterone (P4) and estradiol-17beta (E2) on the efficiency of canine oocyte maturation in vitro is recognized, but little is known about the influence of both steroids on the expression of zona pellucida (ZP) glycoproteins. It has been shown that E2 and P4 used in the IVC significantly influenced canine oocytes meiotic competence, although the effect is specifically related to the combination of hormones used in the experiment. Because both of these steroids may stimulate or inhibit maturation competence of oocytes in a dose-dependent manner, there is a high possibility that they also influence the fertilization ability of canine oocytes. Our study was aimed to analyze whether genes, encoding ZP glycoproteins, are regulated by P4 or E2. Canine cumulus oocyte complexes (COCs) were recovered from anestrous mongrel bitches after ovariohysterectomy and cultured in serum-free tissue culture medium 199. The expression pattern of ZP glycoproteins 2 and 3 (ZP2 and ZP3) mRNAs, using quantitative real-time polymerase chain reaction (RQ-PCR), and of ZP3 and ZP4 proteins, using Western blot analyses, was examined in oocytes after the supplementation of the culture medium with (1) 0.5 μg/mL, 1.0 μg/mL, and 2.0 μg/mL of P4 (experiment 1), or with (2) 2.0 μg/mL E2, and with (3) a combination of E2 (2.0 μg/mL) and P4 (0.5, 1.0, or 2.0 μg/mL, respectively; experiment 2). The analysis revealed an inhibited expression of ZP2 mRNA in oocytes after in vitro maturation (IVM) with different P4 supplementations as compared with oocytes before IVM. The expression of ZP3 mRNA was stimulated (P < 0.01) by the supplementation of 1.0 μg/mL P4. The expression of both ZP3 and ZP4 proteins was also stimulated after the treatment with 1.0 μg/mL P4. On the other hand, the level of ZP2 mRNA was inhibited (P < 0.01) after the supplementation with E2 or with combinations of E2 and P4 as compared with control oocytes. The expression of ZP3 mRNA was significantly higher after the supplementation with E2 and 0.5 μg/mL P4. Similarly, ZP3 and ZP4 proteins were highly expressed (P < 0.01) after such hormone supplementation. The results clearly show that in vitro, P4 regulates the expression of ZP glycoproteins in a dose-dependent manner. We demonstrated that E2 used alone and in combination with P4 upregulates the expression of ZP3 mRNA as well as ZP3 and ZP4 protein in canine oocytes. ZP2 mRNA is downregulated by E2 alone and in combination with E2 and P4. Furthermore, ZP glycoproteins expression is regulated by E2 alone or in combination with P4, and such synergistic or adverse effect is P4 concentration-dependent.
In vitro embryo production in cats and dogs still presents some challenges, and it needs to be optimized to transfer efficient protocols to related wild, endangered species. While the chemical composition of culture media has been the focus of several studies, the importance of culture substrates for oocyte and embryo culture has often been neglected. Traditional in vitro systems, i.e., two-dimensional cultures, do not resemble the physiological environments where cells develop, and they may cause morphological and functional alterations to oocytes and embryos. More modern three-dimensional and microfluidic culture system better mimic the structure and the stimuli found in in vivo conditions, and they could better support the development of oocytes and embryos in vitro, as well as the maintenance of more physiological behaviors. This review describes the different culture systems tested for domestic carnivore reproductive cells along the years, and it summarizes their effects on cultured cells with the purpose of analyzing innovative options to improve in vitro embryo production outcomes.
Dog is the first pet animal since foundation of human civilization. In modern era of human empire, the spending on pets and their care has increased. Assisted reproduction, originally confined to accepted breeds, has now extended to non-domestic canids. Cryopreservation of semen, oocytes and embryos, in vitro fertilization and somatic cell nuclear transfer cloning are used to conserve and propagate valuable genotypes. Stem cells are used as regenerative medicine or clinical health applications.
This chapter presents important aspects of the domestic dog as a valuable research tool for human reproduction with specific focus on canine reproductive biology, and also describes the assisted reproductive technologies available for the dog. It focuses on the dog in modern human society, as well as dog origins and genome, and addresses reproductive biology and assisted reproduction in the dog. The chapter details dog reproduction focusing on reproductive anatomy and physiology, the reproductive cycle and gamete biology. It talks about both external and internal genital organs of male and female dogs. The chapter describes each male reproductive structure of the dog and specific features of dog spermatozoa. Several important breakthroughs have been achieved with the dog, including technologies of assisted reproduction such as in vitro fertilization (IVF), embryo transfer, and somatic cell nuclear transfer (SCNT).
The biological techniques in reproduction began to be developed in the 70s. After laboratory animals and human beings, the success of this technological breakthrough was applied to production-type animals. The first tests in dogs were carried our in 1976, bur in vitro fertilization has not as yet been fully apprehended, and the number of embryos which actually develop is still very, small. The reasons for this slowness to develop are basically related to the particular anatomic and physiological characteristics of the the bitch. The conditions required for oocyte maturation are indeed difficult to reproduce in vitro, In addition, in this type of research there is no financial incentive. Nonetheless, the scientific benefits to be derived from in vitro fertilization in dogs are unquestionable, and its development could lead to an improved control of fertility in both the female (superovulation) and the male (in vitro fertilization test).
The meeting and the fusion of gametes represents the most important event in sexual reproduction. In vitro fertilisation can be used to help in the understanding of the mechanisms and the analysis of the different parameters involved in this process. Unfortunately, in Canidae this technique does not yet allow the complete development of the whole process to occur. In this article, the authors describe the different steps in fertilisation and indicate what is known in Canidae.
Reproductive biotechniques are important for biodiversity preservation and for basic research. The interest for development and application of such biotechniques in dog breeding is steadily increasing and filogenetic similarities enable extrapolation of studies in domestic canidae to wild canidae at risk of extinction, for conservation purposes. This work provides an overview of the main biotechniques employed in domestic dog reproduction. The advances in the development of such biotechniques will contribute to improve dog reproduction and for their application in wild canidae.
The efficiency of the in vitro maturation of canine oocytes is still relatively small. The periovulation period in dogs is different from that in other animal species. This study presents the results of research on the use of various media and substances, such as serum and serum albumin, hormones, energy substrates, antioxidants, antibiotics, antimycotics, and other compounds supporting the maturation of oocytes in vitro. It also describes the methods of culturing oocytes (incubation in a drop, incubation with somatic cells, incubation in the isolated oviduct and in isolated follicles). If the research on conditions simulating the oviduct environment is continued, it may become possible to obtain a higher number of oocytes capable of fertilization than at the present time.
L’ovocyte de chienne constitue un modèle de méiose très particulier parmi les mammifères. Sa maturation
in vitro, étudiée depuis une dizaine d’années seulement, reste très mal maîtrisée : faible taux de métaphase II (10 à 20 % contre plus de 90 % chez les bovins) et fort taux de dégénérescence en culture (20 à 60 %) malgré les essais d’amélioration des milieux de culture. Or la maturation in vitro
est une étape indispensable pour avoir accès, tant chez le chien que chez les Canidés en voie de disparition, aux biotechnologies de la reproduction (fécondation et production d’embryons in vitro notamment). Il est indispensable de mieux comprendre la biologie de l’ovocyte chez la chienne pour améliorer les taux de maturation. Nous nous sommes intéressées, en premier lieu, au rôle de l’AMPc dans la reprise de la méiose in vitro. Nous avons modulé la concentration intraovocytaire d’AMPc en soumettant les ovocytes à des molécules qui la diminuent (Rp-AMPc) ou l’augmentent (dbAMPc et forskoline), ou en dénudant l’ovocyte pour arrêter tout apport par les cellules du cumulus. Nous avons ainsi montré que l’AMPc jouerait un rôle dans la poursuite de la méiose dont la reprise serait également contrôlée par une autre voie, peut-être calcique. En parallèle, nous explorons l’évolution de l’ultrastructure de l’ovocyte au cours de la maturation in vivo et in vitro, pour détecter les anomalies cytoplasmiques qui peuvent apparaître au cours de la maturation.
Canine reproduction has several distinctive features. Firstly, folliculogenesis is unusual as numerous ovarian follicles contain several oocytes (polyovular follicles). Secondly, unlike in other mammalian species, oocytes at the time of ovulation are still at an immature stage (prophase I, germinal vesicle stage), and complete their maturation in the oviduct. This phenomenon is not easy to observe because the canine oocyte has a high lipid content and its DNA is difficult to visualise. Fertilization of immature oocytes has been observed in vitro, however in vivo, fertilization occurs in oocytes at the metaphase II stage, approximately 50 hours after ovulation. The 2-pronuclei stage is reached 72-124 hours after ovulation, and 2-cell embryos are present 96-168 hours after ovulation. The oviductal phase is long and embryos enter the uterine cavity at the morula or early blastocyst stage 10-12 days following ovulation. Implantation occurs 18 to 21 days after ovulation. In spite of all these specificities, studies on canine reproduction were so far mainly clinical. However, current research is focusing on fundamental knowledge, namely the mechanisms controlling oocyte maturation in vivo, in the hope to improve the yield of oocyte maturation in vitro, which is still very low.
• The reproductive cycle in bitches is characterised by four distinct periods (anoestrus, pro-oestrus, oestrus, and metoestrus/dioestrus). The photoperiod and prolactin affects the duration of anoestrus. The growth of ovarian follicles during pro-oestrus is associated with secretion of oestrogens, mainly 17 beta-oestradiol. Ovulation occurs about 30 to 48 hours after peak LH concentration. When the luteal cells start to secret progesterone, there is a rapid increase in blood-progesterone concentration. In the pregnant bitch, it is imperative that there is a rapid fall in progesterone concentration for induction of parturition. Diagnosis of pregnancy is by relaxin assay. Prolactin is the main luteotropic agent from the second month of pregnancy. In male dogs, puberty occurs at different ages depending on the breed and hereditary factors. Spermatozoa must undergo a complex maturation process in the uterus to become fertile. A male dog secretes insufficient concentrations of oestradiol or progesterone to be detectable on routine hormonal assays (3 figures, 2 boxes, 21 references).
The field of cytogenetics is concerned with the study of the structure and properties of chromosomes, their behavior during mitosis and meiosis in reproduction and their influence on the phenotype of the organism. The word "chromosome" was introduced from the Greek language meaning "colored body". Quality of oocytes is one of the important factors affecting the successful rate of in vitro maturation (IVM) and in vitro fertilization (IVF) techniques. The occurrence of chromosomal aberrations in oocytes matured in vitro is an important factor affecting the in vitro production of embryos. Oocytes meiosis is very sensitive to endogenous or exogenous factors, which could lead to chromosomally abnormal oocytes. Abnormalities occurring during gametogenesis and the first stages of development play a significant role in infertility, in vitro fertilization failure and fetal loss. The following areas of cytogenetics will be discussed in more detail; meiosis in female, factors affecting meiotic maturation, chromosome abnormalities in oocytes as diploidy and aneuploidy.
In vitro embryo production has not been confidently applied to the dog successfully. Up to date, only one blactocyst have been achieved by in vitro culture. Therefore, the aim of the present study was to examine the effects of different oocyte diameters (<= 100 mu m and >100 mu m) on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of immature dog oocytes. The study was performed in two steps. At the first step (experiment I), effects of two different oocyte diameters on IVM of dog oocytes were investigated. The nuclear maturation rates were evaluated by aceto-orcein staining method at the end of the IVM. At the second step (experiment II), in vitro matured oocytes were fertilized with fresh spermatozoa for 24 h and in vitro cultured for 7 d. At the end of the IVC period, embryonic development was assessed by microscopic observation at 24 h intervals and fixed for staining by aceto-orcein staining method after 7 days. In comparison relating in IVM and IVF rates, larger oocytes have higher maturation (P<0.05) and cleavage (P<0.01) rates than the smaller ones. Unfortunately, none of the oocyte was reached to morula or blactocyst stage in both groups. In conclusion, it is demonstrated that the oocyte diameter may be a helpful selection criteria for dog in vitro embryo production.
This study was conducted to determine the diameter of canine oocytes that are able to attain full meiotic competence and sperm penetration. Oocytes were collected from ovaries of bitches at various stages of the estrous cycle. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and were divided into four groups based on diameter: <100, 100 to <110, 110 to <120 and ≥120 μm. Following in vitro maturation or fertilization, oocytes were stained to assess nuclear maturation and penetration rates. The mean oocyte diameter was 108.5 ± 0.4 μm. The oocytes displayed size-related ability to undergo meiotic maturation. After culture for 72 h, the rates of oocytes that remained at the germinal vesicle stage in the <110 μm groups were significantly higher (P<0.01) than in the ≥110 μm groups. None of the oocytes <110 μm reached metaphase II (MII), but 4.9 and 21.5% of the oocytes that were greater than 110 and 120 μm, respectively, progressed to MII. After in vitro fertilization for 20 h, 10 to 25% of oocytes were penetrated by spermatozoa, but there were no clear relationships between oocyte diameter and penetration rates of the oocyte by sperm. In the <120 μm groups, sperm penetration was mostly found in oocytes arrested at the germinal vesicle stage. However, a total of eight oocytes ≥120 μm in diameter were penetrated by spermatozoa, of which five oocytes reached MII. These results suggest that there is a clear relationship between oocyte diameter and meiotic competence, but no relationship between oocyte diameter and sperm penetration. Canine oocytes may have acquired meiotic competence once they reach at a diameter of 120 μm, but the oocytes may allow the entry of spermatozoa into the ooplasm irrespective of oocyte diameter.
Some similarities and differences in the ways in which mammalian embryos develop up to the blastocyst stage are described and related, speculatively, to the phylogenetic position of the mammals. Particular attention is given to the farm species and to contrasts between embryogenesis in eutherian and metatherian mammals. The practical implications of these contrasts are discussed in relation to embryo manipulation and transfer. It is argued that comparative studies of mammalian embryogenesis have much to learn from, and much to contribute to, the broader field of developmental biology, and that progress in that field will expand the future applications of farm animal embryo work.
Introduction An Overview of Preimplantation Mammalian Embryology The Sensitivity of Mammalian Preimplantation Embryos to Cooling The Development of Cryopreservation Procedures for Domestic Animal Embryos Summary Bibliography
To identify the time characteristics of ovulation, 5 bitches were evaluated during heat by ultrasonography, laparoscopy and hormonal assay. Blood collections for progesterone and oestradiol 17-β assys and ultrasonography were performed every day and laparoscopy every other day. At ultrasonography, ovaries appeared as anechoic structures about 5 days before the estimated LH peak and gradually increased in size. The greatest changes were observed between days 2 and 4 post-LH peak: echogenicity varied greatly from one animal to another and from one day to the following going from totally anechoic to mixed hypo and hyperechogenicity. Then from day 6, ovaries always appeared as hypoechoic structures assimilated to corpora lutea. At laproscopy, small follicies were seen as early as day 10 before the LH peak. Their size slowly increased to become large protubering follicies around the day of LH peak (day 0). At day 1, corpra luta were observed for the first time and were present in all animals by day 5. During that period preceeding day 5, some ovaries had both corpora lutea and follicies clearly visible on their surface. In one animal, haemorrhagic foci were observed at day 3. Neither ultrasonography, nor laparoscopy allowed precise determination of the time of ovulation. Indeed, follicle collapses was never observed, but changes in echogenicity and in the appearance of the ovaries observed by laparoscopy, suggested that ovulation occurred between days 2 and 4 when progestrone concentrations were 12.6 ± 6.2 and 32.1 ± 10.9 nmol/L, respectively.
The present study investigated the effects of bovine granulosa cell monolayers (BGML) and canine granulosa cell monolayers (CGML) on nuclear maturation of canine oocytes with and without cumulus cells. Cumulus-oocyte complexes (COCs) or cumulus-free oocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM, control group), DMEM with BGML (BGML group), or DMEM with CGML (CGML group) for 72 h at 38.5 °C in 5% CO(2), 5% O(2,) and 90% N(2). All media were supplemented with 10% of FCS, 50 ng/mL of EGF, 2 μg/mL of estradiol-17β, 0.1 IU/mL of hCG, 0.1 IU/mL of FSH, 0.25 mM of pyruvic acid, 100 μM of β-mercaptoethanol, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. In cumulus-enclosed oocytes retrieved from ovaries at estrus and/or diestrus, the highest percentage of M-II oocytes (P < 0.05) was present in the BGML group (27.0%) compared with the CGML group (7.9%) and the control group (3.5%). In cumulus-free oocytes collected from ovaries at estrus and/or diestrus, the proportions of M-II oocytes co-cultured with the CGML were low (3.0%) and similar (P > 0.05) to proportions achieved with control (3.0%). However, the presence of BGML improved (P < 0.05) the ability of denuded oocytes to develop into M-II (10.2%). The BGML group had the highest overall meiotic resumption (P < 0.05), and least oocyte degeneration (P < 0.05) among experimental groups. In conclusion, BGML had a positive impact on the in vitro maturation system, as well as meiotic resumption of canine oocytes.
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