Follicular and oocyte development in gilts of different age

The aim of the present study was to estimate follicular and oocyte development of the same gilts in three phases of their reproductive life--prepuberal gilt (6 months old), cycling gilt (9.5 months old) and primiparous sow. Follicular development was induced by injections of 1000 IU PMSG followed by 500 IU hCG 72 h later. Cumulus-oocyte-complexes (COCs) were recovered from preovulatory follicles of the left ovary, and follicular fluid (FF) from the right ovary always 34 h after hCG by endoscopy. Altogether, 19 gilts were used in the prepuberal (P) and cycling (C) trials and 12 of them in the primiparous trial (S). Altogether 168, 190 and 82 follicles were aspirated from the left ovary and 106, 125 and 42 COCs recovered (recovery rate 60.5 +/- 26.9, 62.7 +/- 20.9 and 52.9 +/- 21.8%). The average number of follicles was higher in C compared to P (19.7 +/- 6.8 vs. 15.7 +/- 6.8, p = 0.06) and to S (14.2 +/- 4.0, p < 0.05), respectively. More uniform expanded COCs were aspirated from prepuberal and cycling gilts as compared to sows (89.7 and 78.4% vs. 46.3%, p < 0.05). Furthermore, the meiotic configuration in oocytes differed (p < 0.05) between these groups (55.5 and 61.7% vs. 0% Telo 1/Meta 2). Concentrations of progesterone in FF decreased (p < 0.05) from 590.0 +/- 333.6 (P) to 249.1 +/- 72.6 (C) and 161.4 +/- 75.2 ng/ml (S). FF concentrations of oestradiol-17 beta were different between gilts and sows (9.3 +/- 2.9, 21.9 +/- 10.6 and 94.0 +/- 15.9 pg/ml, p < 0.05). The progesterone/oestradiol ratio was 72.1, 15.2 and 4.7. Results indicate a different follicular and oocyte development during the investigated lifetime periods. Cycling gilts should preferably be used in IVF and breeding programs. The lower reproductive potential of primiparous sows is taken into consideration at breeding. Prediction of lifetime performance based on individual ovarian reaction of prepuberal gilts is unsuitable.     

Ovarian activity and oocyte development during follicular development in pigs at different reproductive phases estimated by the repeated endoscopic method

The aim of the present study was to assess follicular and oocyte development in the same gilts during three phases of their reproductive life [prepuberal gilts (PP; 6.0 months of age), puberal gilts (P; 9.5 months of age) and primiparous sows (S)]. Follicular development was stimulated by the injection of 1,000 IU of equine chorionic gonadotropin (eCG) followed by 500 IU of human chorionic gonadotropin (hCG) 72 h later. Cumulus-oocyte-complexes (COCs) were recovered by endoscopic ovum pick up/aspiration from preovulatory follicles of the left ovary, and the follicular fluid (FF) from the right ovary was collected 34 h after the hCG treatment by endoscopy. Altogether, 19 pigs were used in the PP and P trials and 12 in the S trial. From the left ovaries, 168, 190 and 82 follicles were aspirated and 106, 125 and 42 COCs, respectively, were recovered (recovery rate 61 +/- 27, 63 +/- 21 and 53 +/- 22%, respectively). The mean number of follicles was greater in the P phase than in the PP phase (19.7 +/- 6.8 vs. 15.7 +/- 6.8; p=0.06) and S phases (14.2 +/- 4.0; p<0.05). More uniform oocytes with an expanded cumulus were aspirated in the P and PP phases than in the S phase (90 and 78 vs. 46%; p<0.05). Furthermore, the meiotic configuration in oocytes (T I/M II stage) differed between the three phases (56 and 62 vs. 0%; p<0.05). Progesterone (P4) levels in FF decreased from 590.0 +/- 333.6 (PP) to 249.1 +/- 72.6 (P) and 161.4 +/- 75.2 ng/ml (S) (p<0.05). Estradiol-17beta (E2) levels differed between PP and P gilts and S sows (9.3 +/- 2.9, 21.9 +/- 10.6 and 94.0 +/- 15.9 pg/ml, respectively; p<0.05), and the P4/E2 ratio was 72, 15 and 5, respectively. These results indicate differences in follicular and oocyte development between the reproductive phases investigated. Puberal gilts should preferably be used in IVF and breeding programs. The lower reproductive potential of primiparous sows must be taken into consideration in breeding. Any prediction of lifetime performance based on individual ovarian reactions of prepuberal gilts is unreliable.     

不同繁殖季节山羊卵母细胞体外成熟及受精效果研究

本试验在2012年11月至2013年5月间进行,笔者从荣昌屠宰场采集了重庆本地黑山羊的卵巢,对于非繁殖季节（冬）和繁殖季节（春）期的卵巢卵母细胞的回收情况作了比较,并观察了不同级别卵母细胞的体外成熟和体外受精情况.结果表明：繁殖季节的卵巢平均回收COCs数、可用COCs数和COCs可用率分别为5.43枚、2.65枚、48.57％,均较非繁殖季节高（P＜0.05）;A、B、C、D级卵母细胞体外成熟率的差异极显著（P＜0.01）,但A、B、C级卵母细胞体外成熟（IVM）卵的体外受精率、卵裂率无显著性差异（P＞0.05）.     

Effect of estrus synchronization with norgestomet on the integrity of oocytes from persistent follicles in beef cattle.

Our objective was to determine whether oocyte integrity is compromised when oocytes are recovered from progestogen-induced persistent follicles. Beef cows were presynchronized using PGF2alpha (PGF). Cows detected in estrus after PGF were assigned to either NOR (one 6-mg norgestomet implant for 10 d starting on d 16 of cycle; day 0 = estrus; n = 112) or CON (control, no implant [n = 128] and presynchronized 8 d later than NOR). All cows received 25 mg of PGF at the end of treatment (NOR, d 26; CON, d 18). Treatments produced persistent preovulatory follicles (NOR) or normal preovulatory-size follicles (CON), which were measured via ultrasonography 1 d before slaughter. Ovaries were collected from all animals (NOR, d 27; CON, d 19) along with random (RAN) ovaries from cattle slaughtered on the same days. Cumulus oocyte complexes (COC) were aspirated from the preovulatory follicles with recovery rates of 63% across treatments. Small follicles (2 to 7 mm diameter) from NOR, CON, and RAN cows were also aspirated to recover COC. Preovulatory follicles were larger (19.5+/-.9 vs. 13.6+/-.4 mm, P<.05), serum P4 was lower (.4+/-.1 vs. 3.9+/-.2 ng/mL, P<.05), and serum E2 was higher (28.7+/-1.6 vs. 7.6+/-.8 pg/mL, P<.05) in NOR than in CON cows. Cumulus oocyte complexes recovered from preovulatory follicles (62 NOR, 64 CON) were matured, fertilized, and cultured in vitro for comparison of embryonic development. A subset (24 NOR, 34 CON) of COC were assigned morphological quality grades. A separate set of recovered COC (10 NOR, 15 CON) was fixed within 1 h after recovery for assessment of the stage of meiosis. Treatments did not differ for oocyte quality grade or stage of meiosis. However, COC from NOR cows had more layers of cumulus cells (P<.05), and more of those COC had undergone cumulus expansion (29.2 vs. 5.9%, P<.05 for NOR vs. CON, respectively). Development of cleaved embryos to the morula and blastocyst stages from preovulatory follicles (22.6% NOR, 18.9% CON) or small follicles (42% NOR, 40% CON, 42% RAN) did not differ with treatment. Oocyte quality and in vitro developmental competence were not compromised for oocytes from induced persistent follicles compared with oocytes from normal preovulatory follicles. Increased expansion of cumulus cells associated with oocytes from progestogen-induced persistent follicles may be relevant to the reduction of in vivo fertility associated with such follicles.     

Preliminary Study in Immature Canine Oocytes Stained with Brilliant Cresyl Blue and Obtained From Bitches with Low and High Progesterone Serum Profiles

This study was conducted: (i) to observe the features and levels of blue colour impregnation in morphologically selected immature canine cumulus oocyte complexes (COCs) stained with the brilliant cresyl blue (BCB) dye, as indicators of quality, and integrity of nuclear oocyte chromatin configuration before in vitro maturation (IVM); (ii) to observe the relationship between the influence of serum progesterone (SP) concentrations from ovary donors and BCB staining of immature dog oocytes. The results showed that out of 138 canine COCs, germinal vesicle (GV) stage prevailed in BCB+ oocytes at percentages of 67.4% (60/89), which were statistically higher than those observed in BCB+/− (52.2%; 23/44) and BCB− (20%; 1/5) oocytes (p = 0.023). Oocytes BCB+ were interpreted as those having completed their growth and therefore possessing the capacity to mature and develop in vitro . Ooplasm and cumulus cells (CCs) of canine oocytes were BCB staining independent. Ooplasm blue colour staining reaction varied between grown oocytes, revealing different levels of glucose-6-phosphate dehydrogenase activity among and within oocytes. Additionally, SP profile of ovary donors was not a relevant indicator for selection of oocytes screened with the BCB stain. Similar numbers of high quality oocytes were observed to be BCB+, BCB+/− and BCB− between groups of females with SP varying from 0 to 2.5 ng/ml (n = 5), and those with SP varying from 2.6 to 16.7 ng/ml (n = 4) (p = 0.680). It may be inferred that bitches with low and high SP profiles have grown oocytes in their ovaries, as determined by the BCB absorbance in their ooplasms.     

Cumulus cell expansion and first polar body extrusion during in vitro oocyte maturation in relation to morphological and morphometric characteristics of the dromedary camel ovary

The morphological and morphometric characteristics of the ovary are fundamental properties for in vitro oocyte maturation. Nuclear maturation, including first polar body (1PB) extrusion, cytoplasmic maturation and cumulus cell (CC) expansion are the criteria for in vitro maturation (IVM) of oocyte. This study was designed to determine the effect of morphological and morphometric features of the ovary on CC expansion and 1PB extrusion during IVM of oocyte in the adult female dromedary camel. The weight, volume and three dimensions of ovaries from slaughtered dromedary camels and oocytes inside zona diameter and zona pellucida thickness were measured. The follicles were classified in regard to the size and oocytes according to their ooplasm appearance and CC compactness. Aspirated cumulus oocyte complexes (COCs) were incubated for 48 hr (with a 6‐hr interval) in Hams‐F10, and CC expansion and 1PB extrusion were assessed. Significant differences were seen in the shape, weight, volume and three dimensions of the ovaries between ≤4‐year‐old and >4‐year‐old dromedary camel (p < .5). Approximately, 95.82% of follicles were 2–4 mm in diameter. The mean (±SD) of inside zona diameter of the oocyte and zona pellucida thickness was 132.22 ± 13.8 and 14.64 ± 2.24 μm, respectively, in >4‐year‐old dromedary camel. The CC expansion and 1PB extrusion were seen in 86% and 21.88% of COCs, respectively. Age and sexual conditions of dromedary camel influence the morphological and morphometric characteristics of the ovary. Most COCs retrieved from 2–6 mm follicles are cultivable. The most slaughterhouse‐derived COCs retrieved from 2–6 mm follicles of non‐pregnant dromedary camels are excellent and good and yielding a most favourable diameter to achieve the developmental competence for IVM in an optimal time of 24–30 hr; the optimal time for CC expansion is 24–30 hr in this species. However, the CC expansion is a prerequisite process, but not sufficient for IVM.     

注射hCG后不同时间猪所排卵泡内COC的形态和卵母细胞减数分裂进程

研究了超排处理注射 h CG后不同时间猪卵丘卵母细胞复合体 (COC)的回收率和形态、卵母细胞减数分裂进程以及 COC形态与生发泡 (GV)染色质构型之间的关系。结果表明 :(1)注射 h CG后 4 h COC的回收率为 5 3.1% ,明显低于注射 h CG后 18、2 2、2 4 h(71.2 %、76 .5 %、70 .0 % ,P<0 .0 5 ) ;(2 )注射 h CG后 18、2 2、2 4 h收集的 COC分别有98.9%、98.0 %、91.4 %的卵丘已经发生扩展 ,而注射 h CG后 4 h收集的 COC则无一发生卵丘扩展 ;(3)注射 h CG后 4h,有少量卵母细胞开始恢复减数分裂 ,至 18h左右开始发生生发泡破裂 (GVBD) ,到 2 2～ 2 4 h有 5 8.4 %～ 6 0 .0 %的卵母细胞发生 GVBD;(4)外层卵丘扩展、放射冠轻微扩展的卵母细胞处于 GV- 1期的比例 (6 9.6 % )明显高于卵丘完全扩展的卵母细胞 (47.8% )和放射冠部分扩展、卵丘已经脱落的卵母细胞 (2 9.3% ) ,而后 2者发生 GVBD的比例(40 %、4 4 % )则略高于前者 (2 7% )。     

休情期银黑狐卵巢形态和卵泡发育的显微结构观察

研究休情期银黑狐卵巢形态和卵泡的显微结构,以揭示银黑狐卵巢发育的一般规律。本试验于2012年12月份采集5只健康一岁龄银黑狐卵巢10枚,用游标卡尺测量其长、宽、厚,用电子天平测量其重量,并对其表面可见卵泡数量进行统计,然后利用光学显微镜对各级卵泡分别观察1~3个,共计原始卵泡30个,初级卵泡20个,次级卵泡15个,三级卵泡12个,成熟卵泡10个,并进行拍照。结果表明:随银黑狐卵巢体积不断增大,其中80%的卵巢重量也随之增大;可见卵泡数量与卵巢体积及重量没有相关性;卵巢由被膜、皮质和髓质构成,髓质位于卵巢内层,分布着较多血管,皮质位于卵巢外层,内有不同发育阶段的卵泡;原始卵泡由卵母细胞和颗粒细胞构成,初级卵泡开始出现透明带物质,到次级卵泡阶段发育完整,三级卵泡出现卵泡腔,卵泡及卵母细胞直径在有腔卵泡阶段比腔前卵泡阶段增长速度快,成熟卵泡的直径及透明带厚度达到最大,各级卵泡均有闭锁现象。     

DNA Fragmentation in Canine Immature Grade 1 Cumulus‐Oocyte Complexes

In this work, we studied the incidence of DNA fragmentation, interpreted as apoptotic changes and assessed by the TUNEL assay, in cumulus cells and oocytes of immature Grade 1 cumulus‐oocyte complexes (COCs) obtained from healthy bitches (n = 27) of three age groups: young (1–3 years; n = 13), adult (4–6 years; n = 8) and elderly (7–10 years; n = 6). Age affected (p < 0.05) Grade 1 COCs recovery rates, with young animals yielding more (p < 0.01) Grade 1 COCs than the other two age groups. Conversely, no differences were observed in the incidence of DNA fragmentation (TUNEL‐positive) in cumulus cells or oocytes between the three age groups. Overall, more than 80% of Grade 1 COCs presented <15% of TUNEL‐positive cumulus cells and enclosed TUNEL‐negative (intact DNA) oocytes. Despite a higher proportion of TUNEL‐negative oocytes being found in the germinal vesicle stage, most of the oocytes with nuclear material compatible with meiosis resumption (MR) or with non‐identifiable nuclear material (ND) did not present DNA fragmentation. No correlation was observed between DNA fragmentations in oocytes and in cumulus cells. We concluded that the morphological parameters used to classify canine Grade 1 COCs are reliable to select a homogeneous population of COCs with low incidence of DNA fragmentation. Furthermore, these results indicate that DNA fragmentation can only explain a minor proportion of the incidence of MR and degeneration in canine oocytes at collection.     

In vitro maturation of oocytes derived from the brown bear (Ursus arctos)

This study was conducted to determine whether meiotic maturation could be induced in ovarian oocytes from the American brown bear (Ursus arctos), a model for gamete "rescue" techniques for endangered ursids. The bears were euthanized, and their ovaries were transported to the laboratory within 4 h. The mean ovarian size was 2.4 x 1.8 cm (range: 2.0-3.3 x 1.5-2.2 cm). The ovaries obtained from the 2 brown bears yielded 97 oocytes (48.5/female), and 88 (90.7%) of them were morphologically classified as normal quality. Oocytes were in vitro matured at 38.5 C in 5% CO2 for 24 or 48 h in TCM-199 supplemented with 10% FBS, 1 microg/ml estradiol-17beta, and 10 microg/ml FSH. In Exp. 1, morphologic evaluation of matured oocytes was conducted by measuring the diameters of oocytes with a zona pellucida (ZP) or cytoplasm without a ZP. In Exp. 2, activation was induced by applying two 20 microsec DC pulses of 2.0 kV/cm delivered by an Electro Cell Fusion Generator. The activated oocytes were cultured in TCM-199 containing 2 mM of 6-dimethylaminopurine for 4 h, in Charles Rosenkrans (CR) 1 for 3 days and the in CR2 for another 4 days. The diameters of the matured bear oocytes with a ZP and with cytoplasm without a ZP (161.8 +/- 6.0 and 135.3 +/- 7.5 microm, respectively) were significantly (P<0.05) larger than those of bovine oocytes (150.7 +/- 4.9 and 118.7 +/- 7.5 microm). The maturation rates of the bear oocytes were 17.6 and 59.4% at 24 and 48 h of in vitro maturation, the percentage of activated oocytes that developed to the 2 or 4-cell stage was 31.6%; however, no blastocysts were observed. These results indicate that bear oocytes can develop to metaphase II in an in vitro culture system and that activated oocytes can develop to the 2 or 4-cell stages.     

The Phosphodiesterase Inhibitor,Isobutyl-1-Methylxanthine Prevents the Sudden Drop in Cyclic Adenosine Monophosphate Concentration and Modulates Glucose Metabolism of Equine Cumulus–Oocyte Complexes Matured in Vitro

Spontaneous nuclear maturation of mammalian oocytes can occur when physically removed from the ovarian follicle during in vitro oocyte maturation (IVM), largely because of a decrease in cyclic adenosine monophosphate (cAMP) concentration. Modulation of oocyte cAMP during IVM by using phosphodiesterase inhibitors has been shown to maintain elevated oocyte cAMP concentrations and control meiotic resumption of bovine and ovine oocytes. This study determined the effect of inclusion of isobutyl-1-methylxanthine (IBMX) during collection and the first 12 hours of incubation of equine oocytes on cAMP concentration and glucose metabolism of cumulus–oocyte complexes (COCs). Abattoir-derived COCs were collected in aspiration medium with (Asp-IBMX) or without (Asp) IBMX. Cumulus–oocyte complexes were then incubated for 12 hours in IVM medium with (Mat-IBMX) or without (Mat) IBMX, followed by additional 24 hours in Mat medium. The cAMP concentration, glucose consumption, lactate production, and metaphase II rates of the COCs were assessed. Cumulus–oocyte complexes aspirated into Asp-IBMX (62.2 ± 2.6 fmol per COC) medium had higher cAMP concentration than Asp (31.8 ± 2.8 fmol per COC) control group (P < .05). Likewise, at 12 hours of IVM, Mat-IBMX group (33.2 ± 2.1 fmol per COC) had higher cAMP concentration than the Mat group (7.68 ± 0.5 fmol per COC; P < .05). Glucose consumption and lactate production were lower during the first 12 hours of incubation in COCs cultured in Mat-IBMX (P < .05). Isobutyl-1-methylxanthine prevented the rapid drop in cAMP concentration and altered metabolism of glucose by the COC. Preventing the sudden drop in cAMP prevents the premature nuclear maturation of in vitro–matured oocytes causing poor developmental competence.     

Infertility in a Dog due to Proximal Cytoplasmic Droplets in the Ejaculate: Investigation of the Significance for Sperm Functionality In Vitro

A 4-year-old Basque Shepherd male dog was presented for breeding soundness evaluation after the dog failed to impregnate the three bitches he had mated. Clinical examination showed no anomaly of the reproductive system. Semen evaluation showed normal sperm count (640 x 10(6)), 80% had progressively motile spermatozoa, and 96% had morphologically abnormal sperm of which 84% had proximal cytoplasmic droplet and 12% had proximal droplet plus other anomaly. A zona pellucida-binding assay, using canine oocytes derived from frozen-thawed ovaries, was performed in order to investigate the zona-binding ability of dog spermatozoa with proximal cytoplasmic droplets. For the zona pellucida-binding assay, ovaries were thawed and minced in phosphate-buffered saline + 0.4% bovine serum albumin, the oocytes recovered were divided into two groups of 35-40 oocytes to be, respectively, used with the infertile dog and with a control fertile dog. Spermatozoa were capacitated in Canine Capacitating Medium (CCM) at 38.5 degrees C and 5% CO(2) in air for 2 h before oocyte insemination. Groups of five to six oocytes placed in 45 microl droplets of CCM were incubated for 1 h. Afterwards, 5 microl of CCM containing 25,000 spermatozoa were added to each droplet and co-incubated for 2 h before fixation and evaluation of the complexes. After oocyte insemination, sperm motility and viability were evaluated: the sample from the infertile dog had 85% sperm motility with fast and linear progressive movement, and sperm viability of 92%. The sample from the control dog showed 40% sperm motility with fast and highly curvilinear and erratic movement, high degree of sperm agglutination and sperm viability of 32%. For the infertile dog the mean number of bound spermatozoa/oocyte was 0.33 whereas for the control dog it was 1.80. It was concluded that dog sperm with proximal cytoplasmic droplets seem to lack normal capacitating ability in vitro, and consequently, they may have reduced capacity to bind to the zona pellucida of canine oocytes.     

Differences in development of bovine oocytes recovered by aspiration or by mincing.

This experiment was performed to clarify relationships between conditions of bovine ovaries and developmental capacity of the follicular oocytes recovered from them and to compare two methods of oocyte collection, aspiration and mincing. Follicular oocytes with surrounding intact, unexpanded cumulus recovered by follicular aspiration or by mincing of tissue from 24 pairs of ovaries were matured and fertilized in vitro. The number of follicular oocytes recovered from pairs of ovaries averaged 32.1 +/- 3.2, but the number recovered varied greatly among the 24 pairs of ovaries (range, 7 to 71). The overall rate of development to the blastocyst stage was 18% (137/771), and the average number of blastocysts produced from a pair of ovaries was 5.7 +/- 1.1 (range, 0 to 17). No relationships were found between the presence of corpora lutea or large follicles and the proportion of oocytes capable of reaching the blastocyst stage in vitro. However, a positive correlation was observed between the number of oocytes obtained from each pair of ovaries and subsequent in vitro development; the correlation was especially high for oocytes obtained by aspiration. These data suggest that the developmental capacity of bovine follicular oocytes after in vitro maturation and fertilization is correlated to the number of antral follicles aspirated from the pair of ovaries.     

A simple method for selection of cumulus-oocyte complexes from bovine ovaries by sedimentation with percoll

Selection of bovine cumulus-oocyte complexes (COCs) for in vitro embryo production (IVP) is generally based on the morphological characteristics of the cumulus cells surrounding the oocytes and the ooplasm under microscopic observation. The purpose of this study was to examine a simple method for selection of COCs by sedimentation with Percoll solutions. COCs were aspirated from ovaries derived from a local slaughterhouse, and the COCs were classified by the morphology of their cumulus cell layers, as follows: Class A, compact and thick; Class B, compact but thin; Class C, partially denuded and thin; and Class D, denuded. Percoll solutions were prepared by diluting Percoll to 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30% solutions, respectively. COCs were placed on the surface of the Percoll solution for 3 min, and the precipitated COCs were transferred to stepwise high density solution. The percentage of Percoll solution just before buoyancy was considered to the specific sedimentary value of the COC and oocyte. The mean sedimentary value of Class A COCs was higher than those of the other classes (P<0.01). The mean sedimentary values of denuded oocytes from Classes A and B were higher than those from Classes C and D (P<0.01). Our results show that sedimentation of COCs and denuded oocytes was generally related to the morphological quality of the COCs, although the sedimentary values ranged widely for one class of COCs and oocytes. The Percoll method can be used for simple selection of COCs.     

Vitrification with DAP 213 and Cryotop of Ex Situ and In Situ Feline Cumulus–Oocyte Complexes

This study was undertaken to compare cryotolerance, in terms of viability and resumption of meiosis after warming and culture (24 and 48 h), of ex situ (isolated) and in situ (enclosed in the ovarian tissue) feline cumulus–oocyte complexes (COCs) vitrified with DAP 213 (2 m DMSO, 1 m acetamide, 3 m propylene glycol) in cryotubes or Cryotop method. Ovaries were harvested from 49 pubertal queens. Of each pair of ovaries, one was dissected to release COCs randomly divided into three groups: fresh COCs (control), ex situ COCs vitrified with DAP 213 and Cryotop. The cortex of the other ovary was sectioned into small fragments (approximately 1.5 mm³) and randomly assigned to be vitrified by DAP 213 or Cryotop. After warming, ex situ and in situ (retrieved form vitrified ovarian tissue) COCs were matured in vitro. Viability of oocytes was highly preserved after warming and culture in all treatments. Proportions of oocytes surrounded by complete layers of viable cumulus cells were remarkably decreased (p < 0.00001) in both vitrification procedures compared to fresh oocytes. Resumption of meiosis occurred in all treatments. After 24 h of culture, results were similar in ex situ and in situ vitrified oocytes regardless of the vitrification protocol used (range 29–40%), albeit lower (p < 0.05) than those of fresh oocytes (65.8%). After 48 h of culture, ex situ oocytes vitrified with Cryotop achieved the rates of meiosis resumption similar to fresh oocytes (53.8% vs 67.5%; p > 0.05) and ex situ and in situ oocytes vitrified with DAP 213 showed similar rates of resumption of meiosis. These findings demonstrated that DAP 213 and Cryotop preserve the viability of ex situ and in situ oocytes, but cumulus cells are highly susceptible to vitrification. However, the capability to resume meiosis evidences that feline immature oocytes vitrified as isolated or enclosed in the ovarian cortex have comparable cryotolerance.     

Follicle formation in the canine ovary after autografting to a peripheral site

This study reports about follicular development on the surface of canine ovarian tissue after autografting under the fascia of the thoracolumbar muscle and about meiotic resumption of follicle-derived oocyte after maturation culture. After ovarian excision from a bitch, each ovary of the pairs was cut approximately into half. The hemi-ovaries were transplanted into the bitch of origin at three different body sites (under the fascia of the quadriceps femoris muscle and the thoracolumbar muscle, and in the deltoid muscle in the scapular region). All grafted ovaries were recovered from the bitch at 35 days post-transplantation. A visible antral follicle was observed on the surface of the ovary grafted under the thoracolumbar fascia. Histological examination revealed viable follicles at different stages of development irrespective of graft site. Most granulosa cells in the follicles at different stages of development expressed proliferating cell nuclear antigen (PCNA). A total of three oocytes were collected from an ovary grafted under the fascia of the thoracolumbar muscle, wherein an oocyte reached metaphase I after maturation culture. This is the first report to demonstrate follicular development and meiotic resumption of oocytes recovered from autografted canine ovarian tissues.     

The effect of GnRH supplement of FSH and PMSG treatments for prepubertal swamp buffalo calves (Bubalus bubalis)

The objective of the experiment was to improve the multifollicle stimulation technique and the ovarian response examination in prepubertal swamp buffalo calves. Six animals were stimulated by gonadotropin hormone 7 days after a progesterone ear-implant. The first stimulation was done by giving 24 mg FSH + 100 microg GnRH (FSH+GnRH) and the second, one month after by giving 2,000 IU PMSG + 100 microg GnRH (PMSG+GnRH). Twenty-four hours after GnRH, the ovarian responses were checked using rectal palpation and real-time B mode ultrasonography. Five out of six animals (83.3%) responded to both treatments and were selected for oocyte collection. The oocytes were aspirated directly following a caudal midline laparotomy. The results of ovarian responses to FSH+GnRH and PMSG+GnRH averaged 17.6+/-12.1 (L-9.8+/-8.7, R=7.8+/-6.2) and 17.4+/-5.6 (L-9.4+/-2.9, R=8.0+/-3.7), respectively. The average number of recovered oocytes per animal was 9.0+/-6.4 and 8.4+/-1.1, respectively which represented a recovery rate of 56.3 (+/- 9.2)% and 51.9 (+/- 10.3)%. More than eighty percent of the recovered oocytes were in an immature stage with more than 2-3 layers of compact cumulus mass. The present study showed that the oocytes were collected successfully in prepubertal buffalo calves after the FSH+GnRH or PMSG+GnRH stimulation and most of the recovered oocytes were immature, which made them suitable for in vitro maturation and fertilization.     

Influence of co‐culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus‐enclosed porcine oocytes in a defined system

Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system.     

牦牛卵巢卵母细胞体外培养成熟条件的建立

研究了不同采集方法和不同培养液对牦牛卵母细胞体外成熟的培养效果。结果:在牦牛乏情期,每个卵巢平均回收卵数为(9.33±4.30),可用卵数为(5.63±4.19)。将牦牛卵丘卵母细胞复合体分别置于5种成熟培养液中培养,成熟率分别为75.56%、71.11%、81.33%、77.33%和77.78%,卵裂率分别为42.22%、33.33%、49.33%、46.67%和42.22%,其中C的成熟液效果最好。来自卵巢表面卵泡的COCs的成熟率(81.33(vs69.33(,P>0.05)高于来自卵巢内卵泡的COCs,卵裂率(49.33%vs34.67%,P<0.05)显著高于来自卵巢内卵泡的COCs。来自明亮卵泡的COCs的成熟率(81.33%vs33.33%,P<0.01)和卵裂率(49.33%vs3.33%,P<0.01)极显著高于来自混浊卵泡的COCs。