Effect of Follicle Size on In Vitro Maturation of Pre‐Pubertal Porcine Cumulus Oocyte Complexes

Very small follicles (<3.0 mm diameter) are over‐represented on the surface of ovaries of non‐cycling pigs, and the oocytes collected from these follicles generally have reduced developmental competence in vitro. This study examined the effect of follicle size on the nuclear maturation (n = 608), the potential of parthenogenetic activation (n = 243) and the cyclic AMP (cAMP) content of pre‐pubertal porcine oocytes (n = 480). In addition, the influence of follicle size on steroid hormone synthesis was analysed. Cumulus oocyte complexes (COCs) flushed from small (2.5–4.0 mm) or large (4.5–6.0 mm) ovarian follicles were cultured for 0, 28 and 46 h. After 46 h of IVM, a greater proportion of oocytes from 4.5‐ to 6.0‐mm follicles reach metaphase II (MII) compared with those from follicles with 2.5–4.0 mm of diameter (96.1 vs 77.0%, respectively; p < 0.001). Parthenogenetic activation of oocytes from large follicles produced higher developmental rates than oocytes from large follicles (p < 0.05). At 28 h, the IVM medium with oocytes from large follicles contained significantly more 17ß‐oestradiol (E₂) than the medium with oocytes from small follicles (5.55 vs 3.45 ng/ml, respectively; p < 0.05) and at 46 h, the medium with oocytes from small follicles contained significantly more progesterone (P₄) than the medium with oocytes from large follicles (276.7 vs 108.2 ng/ml, respectively, p < 0.05). Porcine oocytes from large follicles have higher nuclear and cytoplasmic maturation capacities, but the differences did not appear to be cAMP‐mediated. Our findings also suggest that COCs from small follicles undergo more intensive luteinization than COCs from large follicles. The results show that oocytes from follicles with a diameter greater than 4.0 mm are more suitable for in vitro studies.     

高产奶牛连续活体采卵及卵母细胞体外受精

在超声波扫描仪的指导下,用双孔型采卵针以 １４．７ ｋ Ｐａ 抽吸压经阴道对 ５ 头高产奶牛分别连续实施 ７ 次活体采卵,每 ７ ｄ １ 次,共采集卵子 ２１４ 个,占可见卵泡数的 ５３．９％ ,每次头均采集卵子 ６．１ 个。卵子经过体外培养、体外受精、体外受精胚体外发育培养,于体外受精后 ４８ ｈ 、１６８ ｈ 统计的卵裂率、囊胚发育率分别为 ７５．２％ 和 ２９．７％ 。研究结果表明,在超声波扫描仪指导下对奶牛连续进行活体采卵是可行的,所得卵子应用体外成熟、体外受精、体外培养技术,可生产用于冷冻或移植的胚胎。     

The in vitro developmental competence of oocytes from juvenile calves is related to follicular diameter

This study investigated the relationship between follicle size (FS) and developmental competence of calf oocytes. Cumulus-oocyte-complexes (COCs) from follicles>8 (L-COCs; n=19), 4-8 (M-COCs; n=54), and 2-3 mm (S-COCs; n=155) were recovered from non-stimulated 1-4 months old dairy calves post mortem and ex vivo (laparoscopy), and in parallel from slaughtered adult cows from follicles of identical size categories [>8 (n=91); 4-8 (n=138); 2-3 mm (n=193)]. Morphologically intact COCs were subjected to in vitro maturation, fertilization, and embryo culture. Cleavage rate (CR; 46 h post-insemination=p.i.), rate of morulae/blastocysts (M/Bl; day 7 p.i.), and blastocysts (Bl; day 9 p.i.) were recorded. FS had no effect on the CR in calves. However, calf L-COCs yielded the highest rates of M/Bl and Bl compared with the two other size categories (P<0.05). In contrast, calf S- and M-COCs gave similar rates of M/Bl, whereas the proportion of Bl was lowest for S-COCs (P<0.05). This was almost identical to findings in cows, except that the CR was highest for L-COCs and M/Bl yields were lowest for S-COCs (P<0.05). There were no differences between calf and cows with regard to CR for the respective FS categories. L-COCs from calves and cows yielded similar rates of M/Bl and Bl, whereas calf S- and M-COCs yielded lower rates of Bl than S- and M-COCs from cows and a lower rate of M/Bl when S-and M-COCs were analyzed as one group (P<0.05). Whereas the CR was similar in calves and cows, calf COCs yielded lower rates of M/Bl and Bl (P<0.05). In conclusion, the results show that the developmental competence of calf oocytes is higher in those derived from follicles larger than 8 mm, and thus are almost equally as competent as cow oocytes derived from follicles of identical size. This suggests that calf oocytes acquire developmental competence within the large follicle, potentially due to a process similar to prematuration of the oocyte in the adult cow. It is proposed that procedures that facilitate prematuration, such as "coasting" following a preceding superstimulation, might increase the developmental competence of calf oocytes.     

牛有腔卵泡卵母细胞体外成熟的研究

试验从屠宰场收集了 4 8头青年黄牛、34头青年水牛的卵巢共 16 4个 ,共回收可用卵母细胞 86 4枚。水牛平均每个卵巢可回收 3.2 2枚可用卵母细胞 ,相当于黄牛 (6 .72枚 )的一半。试验结果表明 :水牛卵巢生长卵泡少 ,卵母细胞产量少、质量差 ;3种采集黄牛卵泡卵母细胞的方法 ,用抽吸加切割法平均从每个卵巢回收的可用卵数 (7.71枚 )都极显著高于抽吸法 (6 .19枚 )和切割法 (6 .4 4枚 ) (P <0 .0 1) ,但是该法手续繁杂 ,适于一次且只能收集少量卵巢时使用 ;在黄牛和水牛卵母细胞成熟培养液中用 0 .3%PVP代替 10 %FBS ,牛卵母细胞的体外成熟率无显著差异 (P >0 .0 5 )。     

In vitro Development of Buffalo Oocytes in Media-containing Fluids from Different Size Class Follicles

Studies were conducted to investigate the effect of supplementation of fluid from different sized class [small (SFF, < 3 mm), medium (MFF, 3-8 mm) and large (LFF, > 8 mm)] of normal and cystic (CFF) ovarian follicles in oocyte culture media on oocyte maturation rate and embryo development in vitro and to test the efficacy of follicular fluid (FF) from different size classes as a whole oocyte maturation medium. Results suggested that FF were capable of developing buffalo oocytes to embryonic stage in vitro although its efficacy was lower than that of serum. Regardless of high maturation rates after in vitro maturation (IVM) in media containing FF or IVM in whole FF, low blastocyst rates were obtained after in vitro fertilization (IVF) and culture of embryos. Follicular fluid from small follicles had significantly (p < 0.05) higher potential of developing buffalo oocytes to embryonic stage in vitro than that from medium and large follicles. Cystic FF was not capable of supporting development of buffalo oocytes in vitro.     

Influence of cysteine in conjunction with growth factors on the development of in vitro-produced bovine embryos

Cysteine supplementation to in vitro maturation (IVM) media of bovine oocytes increases cellular glutathione production. Beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mm cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/ml); EGF (10 ng/ml); and IGF-I + EGF (100 + 10 ng/ml). Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the maturation media (12 h C IGF-I + EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I + EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in embryos was assessed. No treatment effect was observed on the expression of these genes. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.     

Maturation competence of swamp buffalo oocytes obtained by ovum pick-up and from slaughterhouse ovaries

This study was designed with the final goal of improving in vitro embryo production in the Thai swamp buffalo (Bubalus bubalis carabensis). Oocytes were collected by ovum pick-up (OPU) from six non-lactating multiparous swamp buffalo twice per week for 10 consecutive sessions followed by once-weekly collection for 10 consecutive sessions without hormone stimulation. In addition, oocytes were collected from slaughterhouse ovaries that were classified as follows: ovaries from non-pregnant cows with a visible corpus luteum (NPCL); pregnant cows with a corpus luteum (P); and non-pregnant cows without a corpus luteum (NP). Follicles in each group of ovaries were categorized as small (2-4 mm), medium-sized (5-8 mm) or large follicles (≥ 9 mm). The quality of the oocytes was assessed by their capacity to undergo in vitro maturation. The total number of observed follicles per session (all sizes combined) was larger in the once-weekly OPU group compared with the twice-weekly OPU group. In particular, the numbers of small and large follicles were higher in the once-weekly OPU group (5.2 ± 0.7 and 0.9 ± 0.2, respectively) than in the twice-weekly OPU group (3.9 ± 0.5 and 0.5 ± 0.1). The number of medium-sized follicles did not differ between the groups. The percentages of oocytes with an abnormal spindle morphology were not different between oocytes from the twice-weekly (30.0%) and the once-weekly (28.6%) OPU groups. A higher percentage of oocytes obtained in vitro (49.5%) exhibited nuclear abnormalities compared with those obtained in vivo (≤34.8%) after in vitro maturation. In conclusion, oocytes can be successfully collected by OPU in the swamp buffalo, without hormonal pretreatment, and per week more good-quality oocytes can be collected by twice-weekly OPU. In addition, oocytes collected from slaughterhouse ovaries can be used with the reproductive status of the cow having no influence on the maturation competence of oocytes.     

Allometric Study on the Relationship between the Growth of Ovarian Follicles and Oocytes in Domestic Cats

The relationship between the growth of preantral and antral follicles and that of their oocytes in ovaries of domestic cats (Felis catus) was analyzed. Eight hundred and five pairs of follicles and oocytes from the ovaries of 51 female cats were collected, and only healthy and fresh follicles and oocytes with or without zona pellucida were used in this study. Immediately after collection, the diameters of follicles and their oocytes were measured. The relationship of the follicle diameter to the oocyte diameter was applied to four regression models and statistically analyzed. The best fitting model was found to be a hyperbolic regression (the coefficient of determination was 0.976 between the follicles and their oocytes with a zona pellucida, y=184x/(x+0.0738); the coefficient of determination was 0.983 between the follicles and their oocytes without a zona pellucida, y=122x/(x+0.0301)). The differentiated equations for the hyperbolic curves in the oocytes with or without a zona pellucida and the follicles were found to be y'=13.6/(x+0.0738)(2) and y'=3.67/(x+0.0301)(2), where y and x were the diameters of the oocytes (μm) and follicles (mm), respectively. When follicles grew to a size larger than 0.4 mm in diameter, the growth rates of their oocytes calculated by the differentiation equations showed an asymptotic depression around zero. Thus, it was suggested that when the follicles grew to a size larger than 0.4 mm in diameter, their oocytes reached full size and ceased to grow and that the zona pellucida stopped growing when the diameter of the follicles reached 0.3 mm in domestic cats.     

Effect of exposure duration of ovaries and oocytes at ambient temperature on parthenogenetic development of porcine follicular oocytes

This study was conducted to evaluate the effects of exposing porcine ovaries to 30-33 C during transportation for 4 h and subsequently room temperature (25 C) for 6 h of storage on in vitro maturation (IVM) and subsequent parthenogenetic development of oocytes collected from the ovaries. After IVM, oocytes having a tight oopalsm membrane and no signs of degeneration were exposed to Dulbecco's phosphate-buffered saline (DPBS) with 7% ethanol (v/v) for 7 min to induce parthenogenetic activation. Moreover, we also determined whether exposure of the collected oocytes to room temperature for 1, 2 and 4 h in DPBS or porcine follicular fluid (pFF) affected parthenogenetic development. When porcine ovaries were stored after transportation, oocytes collected from the stored ovaries showed a significantly higher rate of degeneration after 65 h of IVM (58.4%) and a significantly lower rate of cleavage after parthenogenetic activation (40.1%) than oocytes collected from ovaries immediately after transportation (38.9% and 47.4%, respectively). However, there was no significant difference in developmental rates to the morula and blastocyst stages between these two groups (14.4% and 14.3%, respectively). The duration of preservation, 1, 2, and 4 h, of oocytes in DPBS did not affect parthenogenetic development. In contrast, when preserved for 4 h in pFF, the developmental rates of the oocytes were significantly decreased. This suggested that some factor(s) in follicular fluid affects the developmental rate of oocytes with the passage of time in ambient conditions. These results suggest that even after 6 h storage of ovaries, oocytes having normal morphology after IVM have the same rate of parthenogenetic development as oocytes collected from ovaries just after 4 h of transportation, except for a lower cleavage rate, and that exposure of oocytes to room temperature for 4 h in DPBS does not affect their parthenogenetic developmental competence.     

Effects of pregnant mare's serum gonadotropin treatment on follicular populations and ovulation rates in prepuberal gilts with two morphologically different ovarian types.

Two experiments were conducted to determine the effects of pregnant mare's serum gonadotropin (PMSG) on follicular populations and ovulation rates in prepuberal gilts with grape-type (GT) and honeycomb-type (HT) ovaries. The follicular populations were determined at 170 d of age (d 0) and 19 d after PMSG (d 19). In Exp. 1, the mean number of macroscopic follicles of Classes 2 and 3 was greater (P less than .05) in GT (n = 11) than in HT (n = 32) ovaries at d 0, whereas the mean number of those of Class 1 was greater (P less than .05) in HT ovaries. At d 19, no difference was observed between the two ovarian types for any class of follicles. The PMSG-induced ovulation rates were comparable between the two ovarian types (8.3 vs 7.9, GT vs HT, respectively; P greater than .10). In Exp. 2, the microscopic follicular populations were determined on right and left ovaries removed, respectively, on d 0 and 19. At d 0, GT ovaries (n = 5) contained a greater number of Class 5 nonatretic (P less than .01) and atretic (P less than .02) follicles than did HT ovaries (n = 5), whereas at d 19 the mean number was not statistically different between the two ovarian types (P greater than .10). In contrast, gilts with HT ovaries contained a greater (P less than .01) number of Class 4 atretic follicles than gilts with GT ovaries at d 0, whereas at d 19 the mean number was not statistically different between the two ovarian types (P greater than .10).(ABSTRACT TRUNCATED AT 250 WORDS)     

共培养系统的体细胞类型和状态对猪胚胎早期发育的影响

经体外成熟、受精和培养获得猪胚胎 ,采用体细胞共培养研究了体细胞类型和状态对胚胎早期发育的影响。取体外成熟的不同直径卵泡 (>5 m m,2～ 5 mm)卵母细胞的卵丘团 (颗粒细胞团 ) ,培养铺层后与猪受精卵共培养 ,组间受精卵的卵裂率和发育能力无显著差异 ;根据卵巢的状况对猪输卵管上皮细胞 (POECs)的状态进行分组 ,受精卵和卵巢表面布满卵泡的 POECs共培养 ,卵裂率显著低于卵巢表面有黄体和 /或红体的 POECs共培养组 (P<0 .0 5 ) ,虽然各组间 3～ 4-细胞的发育率无显著差异 ,但卵巢表面有红体和卵泡的 POECs共培养组的 >4-细胞的发育率显著高于卵巢表面布满卵泡的 POECs共培养组 (P<0 .0 5 ) ;受精卵在共培养系统和非共培养系统中的卵裂率无显著差异 ,受精卵非共培养系统中 3～ 4-细胞的发育能力显著低于颗粒细胞共培养组 (P<0 .0 5 ) ,极显著低于 POECs共培养组 (P<0 .0 1) ,无能力突破 4-细胞继续发育 ,与颗粒细胞单层、POECs单层共培养的受精卵 >4-细胞的发育率分别为 2 4.0 %、5 3.8% ,差异显著 (P<0 .0 5 )。结果表明 ,共培养系统对胚胎体外发育的作用 ,一方面与体细胞类型有关 ,另一方面也受输卵管上皮细胞状态的影响     

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.     

Comparison of long-term progestin-based protocols to synchronize estrus before fixed-time artificial insemination in beef heifers

Two experiments were conducted to compare pregnancy rates resulting from fixed-time AI (FTAI) after administration of 1 of 2 long-term controlled internal drug release (CIDR)-based protocols. Heifers were assigned to treatment by age, BW, and pubertal status. The CIDR Select-treated heifers (Exp. 1, n = 37; Exp. 2, n = 192) received a CIDR (1.38 g of progesterone) from d 0 to 14, followed by 100 μg of GnRH, intramuscularly (i.m.) 9 d after CIDR removal (d 23) and PGF(2α) (25 mg, i.m.) 7 d after GnRH treatment (d 30). Heifers assigned to the Show-Me-Synch protocol (Exp. 1, n = 40; Exp. 2, n = 200) received a CIDR from d 0 to 14, followed by PGF(2α) 16 d later (d 30). Artificial insemination was performed at 72 or 66 h after PGF(2α) treatment for the CIDR Select- and Show-Me-Synch-treated heifers, respectively, and each heifer was given GnRH (100 μg, i.m.) at the time of AI. In Exp. 1, ovaries of each heifer were examined by transrectal ultrasonography on d 23 and 30 to characterize follicular dynamics. Follicles ≥5 mm and the presence of corpora lutea were recorded. On d 25, ovaries of each heifer were examined to characterize the status of dominant follicles recorded on d 23. Heifers were fitted with HeatWatch (DDx Inc., Denver, CO) estrus-detection transmitters at PGF(2α) to characterize estrus distribution up to FTAI. The diameter of dominant follicles on d 23 at PGF(2α) and on d 30, and the estrous response after PGF(2α) treatment up to the point of FTAI did not differ between CIDR Select- and Show-Me-Synch-treated heifers. Concentrations of progesterone in serum at PGF(2α) were greater (P = 0.07) in Show-Me-Synch- than CIDR Select-treated heifers (6.0 vs. 4.8 ng/mL, respectively). Pregnancy rates of heifers resulting from FTAI did not differ (P = 0.33) between CIDR Select- and Show-Me-Synch-treated heifers (CIDR Select, 59%; Show-Me-Synch, 70%). In Exp. 2, FTAI pregnancy rates tended (P = 0.07) to be greater in Show-Me-Synch-treated (62%) than in CIDR Select-treated (51%) heifers. Pregnancy rates at the end of the breeding season did not differ (P = 0.72; CIDR Select, 85%; Show-Me-Synch, 83%) between treatments. In summary, pregnancy rates resulting from FTAI were comparable for heifers assigned to each of the 2 long-term progestin-based protocols. The reduced treatment cost and animal handling associated with administration of the Show-Me-Synch protocol offer distinct advantages over the CIDR Select protocol despite similarities in pregnancy rates resulting from FTAI.     

Allometric study on the relation between the growth of preantral and antral follicles and that of oocytes in bovine ovaries

We examined the relation between the growth of preantral and antral follicles and that of their oocytes in the ovaries of Holstein cows. We recovered follicles and oocytes (419 pairs) from the ovaries of 61 cows, and examined the relative growth relating the follicle diameter to the oocyte diameter by using six regression models for only healthy oocytes and all the oocytes including degenerated ones with and/or without zona pellucida. The best fitting model was found to be a hyperbolic regression (R(2): 0.999). The differentiated equation for the hyperbolic curve in normal oocytes with zona pellucida and the follicles was found to be y'=41.0/(x+0.253) (2): y and x are diameters of oocytes (microm) and follicles (mm), respectively. When follicles grew more than 4.0 mm in diameter, the growth rate of the oocytes calculated by the differentiation equation was found to be an asymptotic depression around zero. Thus, it is suggested that when the follicles grow more than 4.0 mm in diameter, the oocytes reach full size and cease to grow. Furthermore, it is considered that the equation can be applied to the assessment of normal growth in oocytes and follicles cultured in vitro.     

Influence of follicle size,methods of retrieval on oocytes yield and morphology in Egyptian Jennies ovaries with special reference to maturation rate in vitro

This work was designed to evaluate the ovarian follicular development, oocytes morphology, methods of oocytes reterival, and the effect of different in vitro maturation (IVM) media on cumulus cell expansion and nuclear maturation of Jennies oocytes. Experiment 1, the number of small (<6 mm), medium (6 to 9 mm) and large size (>10 mm) ovarian follicles was recorded. Cumulus-oocyte-complexes (COCs) were reterived and classified into 4 Grades based on their cumulus-cells investment and the homogenous of the ooplasm. In Experiment 2, COCs were recovered by using 18-G, 20-G needle or slicing and scraping of ovarian follicles to determine the number and morphology of the recovered COCs. In Experiment 3, Grade A and B COCs were IVM in DMEM-HG, DMEM-LG, DMEM-F12, TCM199, TCM199-F12 or CR1aa media supplemented with 10 % FCS?+?10 μg FSH/mL?+?10 IU hCG/mL?+?50 μg/mL gentamicin. Maturation was performed for 36 h at 38.5 °C under 5 % CO₂ in humidified air. After IVM, cumulus cell expansion and oocytes nuclear canfiguration were determined. An average of 6.40?±?0.26 follicles was recorded per Jenny ovary, representing 3.37?±?0.46, 1.89?±?0.14 and 1.14?±?0.16, for the small, medium and large size follicles, respectively. Oocyte recovery was higher (P?P?P?P?P?P?P?P?Conclusion: Slicing and scraping or aspiration of follicles using 18-G needle increased the number and percentage of Grade A Jennies oocytes. TCM199-F12, CR1aa and TCM199 medi are more suitable for IVM of Jenny oocytes by promoting cumulus cells expansion and nuclear maturation to M II stage.     

Attempt at intracytoplasmic sperm injection of in vitro matured oocytes in common minke whales (Balaenoptera acutorostrata) captured during the Kushiro Coast Survey

The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; P<0.01). In Experiment 2, a total of 35 and 46 immature oocytes derived from adult (n=2) and prepubertal (n=6) minke whales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prepubertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.     

输卵管和颗粒细胞单层对牛体外受精胚胎发育的影响

以屠宰场牛卵巢为试验材料,研究输卵管细胞单层(OCM)和颗粒细胞单层(GCM)对牛卵母细胞体外成熟(IVM)、体外受精(IVF)和体外培养(IVC)后胚胎发育能力的影响。(1)从卵泡抽取卵丘卵母细胞复合体(COCs),并根据卵母细胞外面卵丘细胞的层数将其分为3类:1级(≥4层);2级(2～3层);3级(0～1层)。作分别在IVM和IVC培养液中添加GCM(1×106个/mL)与不添加的对比试验。结果显示:添加GCM对1级卵母细胞的卵裂率、6～8细胞发育率和囊胚率无明显影响(P>0.05);但添加GCM的2级、3级卵母细胞,受精后的卵裂率、6～8细胞发育率和囊胚率分别高于未添加组(P<0.05)。(2)所有卵母细胞(包括COCs和裸卵)被随机分为3个组,在其IVM和IVC培养液中分别添加OCM、GCM或不添加体细胞(对照组)。结果显示:OCM和GCM组的卵裂率、6～8细胞发育率和囊胚率均高于对照组(P<0.05),而两试验组之间差异不显著。     

Effects of gonadotropin treatment on ovarian follicle growth and granulosal cell aromatase activity in prepuberal gilts

This experiment was conducted to compare the ability of USDA porcine FSH-B-1 (pFSH), USDA porcine LH-B-1 (pLH), and pregnant mare's serum gonadotropin (PMSG) to grow large follicles and induce granulosal cell aromatase activity in prepuberal gilts. Twenty-four gilts (164 d old) received one of four treatments by i.m. injection: 1) saline once, n = 8; 2) pFSH (8 micrograms/kg BW, nine times at 8-h intervals), n = 5; 3) pLH (2 micrograms/kg BW, nine times at 8-h intervals), n = 6; or 4) PMSG (15 IU/kg BW, once), n = 5. At slaughter, 72 h after the first injection, the ovaries to saline-treated gilts contained an average of 104 surface antral follicles 1 to 3 mm in diameter. compared to treatment with saline, pFSH increased (P less than .05) the number of follicles 46%, whereas pLH or PMSG decreased (P less than .05) the number by 70 and 84%, respectively. Compared with saline, treatment with PMSG or pLH induced growth of large follicles (7 to 9 mm) (10.8 and 4.8 follicles/gilt, respectively), increased plasma estrogen, increased granulosal cell aromatase activity, and decreased plasma FSH by 51 and 69%; treatment with pFSH had no significant effect on these traits. Results indicate that injected pFSH did not cause growth of large follicles or induce granulosal cell aromatase activity in prepuberal gilts. In contrast, LH initiated growth and increased granulosal cell aromatase activity in a small number of follicles and accelerated atresia among the remaining follicles.     

The endothelial nitric oxide synthase/nitric oxide system is involved in the defective quality of bovine oocytes from low mid-antral follicle count ovaries

In a previous survey concerning cows of reproductive age, we demonstrated that oocytes isolated from ovaries with <10 medium antral follicles of 2 to 6 mm in diameter (low ovaries; Lo) show less developmental competence than oocytes collected from ovaries with >10 medium antral follicles (high ovaries; Hi). The aim of the present study was to evaluate whether a defective endothelial nitric oxide synthase/nitric oxide (eNOS/NO) system and vasculature in healthy medium antral follicles is likely to reduce oocyte competence from Lo ovaries. Thus, experiments were conducted to 1) immunolocalize eNOS protein during folliculogenesis; 2) quantify eNOS protein/vasculature in the follicle wall; and 3) verify if NO donor, S-nitroso acetyl penicillamine (SNAP) administration during in vitro maturation affects developmental competence of oocytes isolated from Lo ovaries. Endothelial nitric oxide synthase protein was detected in granulosa and theca cells, as well as in blood vessels from primordial to antral follicles. Quantitative analysis indicated that in medium antral follicles from Lo ovaries, eNOS protein expression and vasculature were reduced (P < 0.05). The addition of SNAP improved blastocyst and hatching rates of oocytes from Lo ovaries, promoting a percentage similar to oocytes from Hi ovaries, and reduced the percentage of apoptotic nuclei in in vitro-produced blastocysts (P < 0.05). Results from our study suggest that in bovine ovaries with small mid antral follicle number, a defective eNOS/NO system is related to a reduced follicle vasculature and may affect oocyte quality, thus inducing a premature decline of fertility.     

AREG对绵羊小腔卵泡卵母细胞体外成熟的影响

旨在研究双调蛋白（AREG）对绵羊小腔卵泡卵母细胞体外成熟（IVM）的影响。本研究从屠宰场绵羊卵巢上采集小腔和中腔卵泡的卵母细胞进行试验，利用自发荧光检测卵母细胞的NAD （P） H和FAD⁺⁺水平，利用JC-1检测卵母细胞的线粒体膜电位；两种来源的卵母细胞经体外成熟后，比较其卵丘扩展指数（CEI）、第一极体排出率（MII%）；比较AREG、AREG+GDF9、AREG+BMP15、AREG+GDF9+BMP15、GDF9+BMP15对小腔卵泡卵母细胞体外成熟后的CEI、MII%及卵母细胞线粒体膜电位的影响；检测了AREG+GDF9+BMP15对小腔卵泡卵母细胞体外成熟后的NAD （P） H和FAD⁺⁺水平及其受精后发育能力的影响。结果表明，成熟前，小腔卵泡卵母细胞的线粒体膜电位和FAD⁺⁺水平均显著低于中腔卵泡卵母细胞（P<0.05）；体外成熟培养后，小腔卵泡卵母细胞的CEI和MII%均显著低于中腔卵泡卵母细胞（P<0.05）。与对照组相比，AREG+GDF9+BMP15显著提高了小腔卵泡卵母细胞体外成熟后的CEI、MII%和线粒体膜电位（P<0.05），且与中腔卵泡卵母细胞组差异不显著（P>0.05）；另外，AREG+GDF9+BMP15显著提高了小腔卵泡卵母细胞体外成熟后的NAD （P） H和FAD⁺⁺水平（P<0.05），且与中腔卵泡卵母细胞组差异不显著（P>0.05）。与对照组相比，在成熟液中添加AREG+GDF9+BMP15可以明显提高小腔卵泡卵母细胞体外受精后的卵裂率和囊胚率（分别为（43.79±3.69）%、（28.54±4.31）%和（78.99±1.12）%、（47.46±2.50）%，P<0.05），而且与中腔卵泡卵母细胞组无显著差异（P>0.05）。综上表明，绵羊小腔卵泡卵母细胞的代谢水平及IVM质量较低， AREG在GDF9和BMP15的协同作用下可以显著提高小腔卵泡卵母细胞的代谢水平及IVM质量，并进一步提高小腔卵泡卵母细胞体外受精后的发育能力。