水牛分离精子与不同来源卵母细胞体外受精的效果研究

本研究的目的是探讨水牛分离精子与不同来源(活体采卵或屠宰场卵巢采卵)卵母细胞体外受精的效果。活体采卵是选用20头空怀河流型母水牛(其中摩拉母牛12头,尼里-拉菲母水牛8头)每间隔3 d采卵1次,连续采卵5~6周,活体采集卵母细胞;屠宰场卵巢采卵是收集屠宰场水牛卵巢,用10 mL注射器连接18 G针头吸取水牛卵巢上可视的卵泡来收集卵母细胞。将收集的AB级水牛卵母细胞在相同的条件下进行体外成熟、然后用分离或未分离精子进行体外受精以及体外培养至囊胚。结果发现:活体采卵组和屠宰场收集的水牛卵母细胞组用分离精子受精分裂率和囊胚率没有差异(P>0.05);分离精子和未分离精子的体外受精分裂率和囊胚率也没有差异(P>0.05)。由此说明,水牛分离精子可以用于体外生产性控胚胎。     

Isolation and Culture of Ovine and Bubaline Small and Large Pre-antral Follicles: Effect of Cyclicity and Presence of a Dominant Follicle

Studies were conducted to examine the effects of the cyclicity and the presence of a dominant follicle (DF) in ovary on the recovery and in vitro growth of pre-antral follicles (PFs) in sheep and buffalo. Small pre-antral follicles (SPFs, 100–250 μm) and large pre-antral follicles (LPFs, 250–450 μm) were isolated from slaughterhouse ovaries in the breeding seasons by a mechanical and enzymatic method. The sheep and buffalo PFs were cultured in vitro for 6 and 15 days, respectively, and examined for their growth, survival and antrum formation rates and growth rates of oocytes in cultured pre-antral follicles. The follicles of the sheep and buffalo were recovered and cultured simultaneously within replicates. The recovery rates (number per ovary) of both SPFs and LPFs were significantly (p < 0.05) higher in cyclic ewes (SPFs: 22.0 ± 3.3 vs 12.1 ± 2.6 and LPFs: 16.0 ± 3.6 vs 9.2 ± 1.8) and buffaloes (SPFs: 9.2 ± 1.3 vs 4.1 ± 1.0 and LPFs: 10.3 ± 2.7 vs 5.4 ± 0.7) compared with those recovered from acyclic ones. Presence of a DF in ovary significantly (p < 0.05) reduced the recovery rates of LPFs in ewes (9.06 ± 2.7 vs 16.4 ± 3.8) but had no effect in buffalo. Cyclicity of animals or follicular dominance had no effects on in vitro growth, survival and antrum formation rates and growth rates of oocytes in cultured PFs of SPFs and LPFs in both sheep and buffalo. The in vitro growth, survival and antrum formation rates of LPFs and growth rates of oocytes in cultured LPFs were significantly (p < 0.05) higher than those observed in SPFs in both sheep and buffalo. The overall recovery and growth rates of the PFs were lower in buffaloes compared with ewes.     

Effects of follicle‐stimulating hormone followed by gonadotropin‐releasing hormone on embryo production by ovum pick‐up and in vitro fertilization in the river buffalo (Bubalus bubalis)

In this study, we examined the effects of superstimulation using follicle‐stimulating hormone (FSH) followed by gonadotropin‐releasing hormone (GnRH) on buffalo embryo production by ultrasound‐guided ovum pick‐up (OPU) and in vitro fertilization (IVF). Nine Murrah buffaloes were subjected to OPU‐IVF without superstimulation (control). The morphologies of the oocytes collected were evaluated, and oocytes were then submitted to in vitro maturation (IVM). Two days after OPU, same nine buffaloes were treated with twice‐daily injections of FSH for 3 days for superstimulation followed by a GnRH injection. Oocytes were collected by OPU 23–24 hr after the GnRH injection and submitted to IVM (the superstimulated group). The total number of follicles, number of follicles with a diameter > 8 mm, and number of oocytes surrounded by multi‐layered cumulus cells were higher in the superstimulated group than in the control group (p ≤ 0.05). After IVF, the percentages of cleavage and development to blastocysts were higher in the superstimulated group than in the control group (p < 0.05). In conclusion, superstimulation improved the quality of oocytes and the embryo productivity of OPU‐IVF in river buffaloes.     

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.     

Anti-Muellerian hormone levels in plasma of Holstein-Friesian heifers as a predictive parameter for ovum pick-up and embryo production outcomes

The aim of this study was to investigate whether plasma anti-Muellerian hormone (AMH) levels of Holstein-Friesian heifers could be used to predict ovum pick-up (OPU) and embryo production outcomes. Plasma samples and data were collected from 64 heifers, which underwent repeated OPU with subsequent in vitro embryo production followed by embryo flushing after superovulation. AMH levels were significantly positively correlated with the number of follicles aspirated per OPU session (r = 0.45), recovered oocytes per OPU (r =0.43) and in vitro produced embryos per OPU (r = 0.28). No significant correlations between AMH and in vivo produced embryos were ascertained. Our results suggest that correlations between AMH and outcomes of an OPU-IVF program are too low to use AMH as a precise predictive parameter for the success of a particular OPU procedure in Holstein-Friesian heifers. However, AMH can help to identify groups of very good or very poor oocyte donors.     

Ovum Pick-up in Cycling and Lactating Postpartum Swamp Buffaloes (Bubalis bubalis)

The objective of this study was to evaluate the efficiency of Ovum Pick Up (OPU) in cycling (n = 5) and lactating, postpartum, swamp buffaloes (n = 6) with and without gonadotropin stimulation. The OPU was performed every two weeks in all groups of animals, for a total of six sessions. Thirty collections were performed in five cycling buffaloes and 36 collections in six lactating postpartum buffaloes. Buffaloes that received hormonal stimulation were given a total of 400 mg, follicle stimulating hormone (FSH), administered twice daily over 3 days in decreasing doses, together with 100 microg of GnRH, 24 h after the last FSH injection. Following a resting period of 1 month, the two groups of buffaloes, were subjected to the same OPU regimen, but without any hormonal treatment for an additional six OPU sessions. The number of aspirated follicles recorded from the hormonal stimulated, cycling animals and lactating, postpartum buffaloes was not significantly different, 7.2 +/- 3.7 and 9.0 +/- 3.2, respectively (p > 0.05). Recovered oocytes collected from the two groups of hormonally stimulated animals were also not statistically different: 3.7 +/- 2.7 in the cycling and 5.9 +/- 3.5 in the lactating postpartum group (p > 0.05). In the two groups of buffaloes not receiving hormonal stimulation, the number of aspirated follicles was not significantly different: 2.1 +/- 1.4 and 1.4 +/- 0.7 in cycling and lactating postpartum buffaloes respectively (p > 0.05). Recovered oocytes in the non-treated groups were also similar: 1.4 +/- 1.3 vs 0.7 +/- 0.8 in cycling and lactating buffaloes (p > 0.05). Among stimulated buffaloes, most aspirated follicles were small in size (< or =5 mm), whereas they were mostly medium and large sizes in the non-treated buffaloes. The oocyte recovery rate in both the groups, cycling and lactating postpartum, were 51.6% and 69.5% in stimulated groups and 55.0% and 53.1% in non-stimulated groups (p > 0.05). The majority of recovered oocytes were single- and multi-layered, and the number was greater in the cycling than in the lactating, postpartum buffaloes. The number and quality of recovered oocytes was similar in all groups of buffaloes whether they were received or did not receive hormonal stimulation. Moreover no difference was found in multi- and single-layered oocytes between cycling and lactating, postpartum buffaloes. In conclusion, OPU can be performed successfully in swamp buffalo in different reproductive status and FSH administration was shown to increase the number of aspirated oocytes in both cycling and lactating, postpartum buffaloes.     

A new method for bovine embryo production: a potential alternative to superovulation

Eight cows were used to study the feasibility of transvaginal ultrasound-guided puncture of follicles as a method for the collection of immature oocytes for embryo production in vitro. In six trials at intervals of seven days, 104 oocytes were collected. After in vitro maturation and fertilisation the 104 oocytes were transferred to the oviducts of sheep. Six days later, 75 oocytes were recovered by flushing the oviducts. Twenty-four per cent of the recovered oocytes/embryos had developed into transferable and viable morulae and, or, blastocysts. The data show that this non-surgical and repeated collection of immature oocytes can be used successfully for the in vitro production of bovine embryos. The procedure may produce yields of embryos comparable to those obtainable by conventional superovulation procedures.     

Repeat Ovum Pick-up and In Vitro Embryo Production from Adult Ewes with and without FSH Treatment

Ovum pick-up (OPU) was performed three times on adult ewes after synchronization with (n = 4) or without (n = 4) FSH treatment to investigate the effects of FSH treatment on the number of ovarian follicles, oocytes recovered, oocyte quality and development in vitro. FSH treatment increased the number of ovarian follicles (85 vs 162) and oocytes recovered (33 vs 91), although recovery rate was similar for ewes with and without FSH (91/162, 56.2% and 33/85, 38.8% respectively). Of the oocytes recovered, those classified as grades I and II were similar between ewes with (78/91, 85.7%) and without FSH treatment (27/33, 81.8%). The number of ovarian follicles was similar after repeated OPU for ewes treated with FSH, but for ewes not treated with FSH the number of ovarian follicles decreased with repeated OPU. The number of oocytes recovered decreased for FSH-treated ewes only, while the oocyte recovery rate and proportion of oocytes classified as grades I and II were not affected by repeated OPU. Oocyte cleavage (46/78, 58.9% and 19/24, 79.2%) and blastocyst formation (35/46, 76.1% and 12/19, 63.2% respectively) were similar for ewes with and without FSH treatment. The number of ovarian follicles varied between ewes (p < 0.05) although the number of oocytes recovered and oocyte development in vitro were similar between ewes.     

Bovine OPU‐Derived Oocytes can be Matured In Vitro for 16–28 h with Similar Developmental Capacity

The aim of this study was to determine the optimal maturation culture period of ovum pick up (OPU)‐derived cumulus oocytes complexes (COCs) in relation to their developmental capacity. Embryo production, embryo cryotolerance, post‐transfer embryonic survival and calf characteristics such as gestation length, birthweight and sex ratio were investigated. This retrospective study covers the analyses of ovum pick up –in vitro production and calving results from a commercial programme that took place between March 1994 and September 2004. Donors were both heifers (of which approximately 90% pregnant) and cows (of which approximately 10% pregnant). Embryo production analyses were based on 7800 OPU sessions conducted from January 1995 until January 1999. Analyses of calving rate were based on 13 468 embryo transfers performed during January 1995 until May 2002. Analyses on calf characteristics were based on 2162 calves born between March 1994 and September 2004. The in vitro maturation culture period ranged from 16 to 28 h. The mean production rate of transferable embryos was 16.5% (1.2 embryos per OPU session). Length of maturation culture period did not affect the production of transferable embryos. Mean calving rate was 40.9% and 38.7% for fresh and frozen/thawed embryos, respectively. Calving rate was not affected by the maturation culture period. Mean birthweight, gestation length and proportion of male calves were 46 kg, 281.9 days and 52.8%, respectively. Maturation culture period did not affect these variables. In conclusion, this study shows that the in vitro maturation culture period within the range of 16–28 h does not affect in vitro embryo production, embryo cryotolerance, post‐transfer embryonic survival and calf characteristics, suggesting that all COC batches collected by OPU on the same day, can be fertilized in one IVF session without a significant loss in the production from oocyte to calf.     

Influence of the FecXR Allele in Heterozygous Ewes on Follicular Population and Outcomes of IVP and ET using LOPU‐Derived Oocytes

Ewes heterozygous for the FecX^R allele (R+) in the bone morphogenetic protein 15 (BMP15) gene display increased ovulation rate and prolificacy. Besides this phenotypic advantage, the influence of the FecX^R allele on follicle number and size, oocyte competence and in vitro production (IVP) remains undefined. With these aims, 8 R+ and 8 wild‐type (++) ewes were subjected to 2 laparoscopic ovum pick‐up (LOPU) trials (four sessions per trial; two with and two without FSH) and subsequent IVP and fresh embryo transfer. All follicles >3 mm were punctured (n = 1673). Genotype did not significantly affect the number of punctured follicles per ewe and session (10.4 and 10.2 in R+ and ++ untreated ewes, 17.4 and 14.3 in R+ and ++ FSH‐treated ewes, respectively), but follicular diameter of R+ ewes was significantly reduced compared with ++ ewes (?0.2 mm in untreated and ?0.8 mm in FSH‐treated ewes; p < 0.01). R+ ewes showed higher recovery rate and increased numbers of total and suitable cumulus–oocyte complexes for in vitro maturation (IVM). Similar rates of day 8 blastocysts were observed in R+ (36.1%, 147/407) and ++ (32.6%, 100/307) ewes, but the final output of day 8 blastocysts per ewe and session was higher in R+ ewes (+0.75; p < 0.005), without differences in survival rate at birth of the transferred embryos (40.4%, 21/52 vs 36.4%, 16/44, respectively). In conclusion, a higher number of oocytes proven to be competent for in vitro development and embryo survival after transfer are recovered from R+ ewes, despite the lower mean size of their follicles at puncture.     

Effects of Ovum Pick-up Frequency and FSH Stimulation: A Retrospective Study on Seven Years of Beef Cattle In Vitro Embryo Production

The aim of this retrospective study was to compare the number of follicles, cumulus oocyte complexes (COCs) and cultured In Vitro Produced (IVP) embryos obtained from 1396 non‐stimulated Ovum Pick‐up (OPU) sessions on 81 donor animals in a twice weekly OPU scheme. Results were obtained from 640 sessions following FSH‐LH superstimulation, on 112 donors subjected to OPU once every 2 weeks. The stimulation protocol started with the insertion of an ear implant containing 3 mg norgestomet (Crestar, Intervet, Belgium) 8 days before puncture (day ?8). The dominant follicle was ablated by ultrasound‐guided follicle puncture on day ?6. On day ?3 and day ?2, cows were injected with FSH (Ovagen, ICP) twice daily (8 am to 8 pm ), i.e. a total dose of 160 μg FSH and 40 μg LG per donor per stimulation cycle. Animals were punctured 48 h after the last FSH injection (day 0). Progesterone implants were removed the next day. Stimulated donor cows were treated with this protocol at 14‐day intervals. Follicles were visualized with a Dynamic Imaging ultrasound scanner, equipped with a 6.5 MHz sectorial probe. Follicles were punctured with 55 cm long, 18 gauge needles at an aspiration pressure corresponding to a flow rate of 15 ml/min. Cumulus oocyte complexes were recovered and processed in a routine IVF set‐up. Results demonstrate that, expressed per session, FSH stimulation prior to OPU increases production efficiency with significantly more follicles punctured and oocytes retrieved. However, when overall results during comparable 2‐week periods are considered (four non‐stimulated sessions vs one stimulated), more follicles are punctured and more oocytes are retrieved using the non‐stimulated protocol. No significant differences in the number of cultured embryos could be detected, indicating that FSH/LH stimulation prior to OPU might have a positive effect on in vitro oocyte developmental competence as more embryos are cultured with less, presumably better‐quality, oocytes.     

Collection of Oocytes Through Transvaginal Ovum Pick-up for In Vitro Embryo Production in Nanyang Yellow Cattle

The objectives of this study were to use ultrasound-guided transvaginal follicular aspiration to develop a technique for preservation of Nanyang Yellow cattle germ plasm. In Nanyang Yellow heifers, aspiration twice per week yielded a higher mean numbers of aspirated follicles, recovered oocytes and viable oocytes per session than once-weekly aspiration. Intriguingly, twice-weekly aspiration from Holstein heifers yielded a higher mean numbers of aspirated follicles, recovered oocytes and viable oocytes per session than in Nanyang Yellow heifers. Moreover, the oocyte recovery rate was significantly higher in Holstein heifers than in Nanyang Yellow heifers. Importantly, similar rates of embryos undergoing cleavage division were observed in Nanyang Yellow and Holstein, as was the number of viable day 8 blastocysts. These observations demonstrate that twice-weekly aspiration harvested the similar proportions of viable oocytes from Nanyang Yellow and Holstein follicles, and that twice-weekly aspiration yields a higher number of viable oocytes than once-weekly aspiration in Nanyang Yellow heifers. The results also suggest that the combination of ultrasound-guided transvaginal follicular aspiration and in vitro production is an effective and practical strategy for establishment of a germ plasm preservation programme for Nanyang Yellow cattle.     

Efficiency of Repeated In Vivo Oocyte and Embryo Recovery After rhFSH Treatment in Rabbits

This study aims to assess the efficiency of in vivo oocyte and embryo recovery after a recombinant human FSH (rhFSH) treatment in rabbit does. Females were distributed in two experimental groups: donor does were treated with rhFSH (superovulation group) for 3 days prior to artificial insemination (embryo recovery) or ovulation induction (oocyte recovery) and does without treatment remained as the control group. Mature oocytes or embryos were collected with the laparoscopy technique 16 h after ovulation induction (oocytes) or 72 h after artificial insemination (embryos). Up to four recoveries were performed with each doe. Recovery efficiencies differed significantly between embryos (84%) and oocytes (58%). Yet, the recovery rates for the superovulation and control groups did not differ. The rhFSH group was associated with a significant increase (p < 0.05) in the number of oocytes and embryos recovered in comparison with the control group (10.2 ± 1.0 and 14.3 ± 1.2 vs 6.0 ± 2.7 and 8.4 ± 2.3 for oocytes and embryos, respectively). Results from this study indicate that repeated in vivo oocyte and embryo recovery from rhFSH superovulated does maximizes the number of oocytes or embryos collected from the same female.     

水牛卵母细胞的体外受精及其胚胎发育的研究

本研究比较了不同来源的水牛卵母细胞的体外受精及其胚胎发育。对10头摩拉母水牛连续6周进行活体采卵,共采集292枚卵母细胞,头均回收卵母细胞4.87枚,头均A级卵数为3.07枚,从屠宰场收集74头水牛卵巢共采集559枚卵母细胞,头均回收卵母细胞和A级卵母细胞分别为7.55枚和5.20枚,均显著高于活体采集的水牛卵母细胞(P<0.01)。将收集的AB级水牛卵母细胞在相同的条件下进行体外成熟、体外受精和体外培养至囊胚。结果表明,活体采卵组和屠宰水牛卵母细胞组受精分裂率差异不显著,分别为54.81%和57.73%。屠宰水牛卵母细胞组的囊胚率则高于活体采卵组(28.78%vs21.34%,P<0.05)。利用两组的胚胎进行常规法冷冻保存,冻胚解冻后的存活率差异不显著(P>0.05)。     

Effect of Days Post-Partum, Breed and Ovum Pick-Up Scheme on Bovine Oocyte Recovery and Embryo Development

The objective of this study was to investigate (i) the effect of two different ovum pick-up (OPU) schemes (once vs twice weekly aspirations) on oocyte recovery rate, quality and subsequent in vitro embryo development, (ii) the influence of days post-partum on oocyte recovery and (iii) possible differences in OPU results from two different herds. In group A, OPU was performed twice weekly in two Holstein Friesian (HF) and three Danish Red and White (DRW) cows from a private herd. In the research herd, two groups of eight HF cows were investigated: group B (OPU once weekly) and group C (OPU twice weekly). The collected oocytes were subsequently submitted to in vitro embryo production. More oocytes were recovered from the private herd when compared with the research herd. In the research herd, the twice weekly scheme aspirated more oocytes than the once weekly scheme. The quality of the retrieved oocytes was significantly different between groups B and C but not between groups A and C, and HF cows yielded higher quality oocytes than DRW cows (p = 0.029). Oocytes from group C showed higher level of embryonic development than group B oocytes. No differences in blastocyst rates were observed between groups A and C. Session affected the number of retrieved oocytes and subsequent developmental rates, with these being lower in the first compared with the last sessions. Finally, there was no significant effect of days post-partum in the number and quality of the retrieved oocytes, likely because of the small group size and high variation between sessions.     

优势卵泡与促卵泡素对牛活体采卵效果的影响

将8头荷期坦供体奶牛随机分为4组,每组2头,分别按优势卵泡+注射促卵泡素(FSH)、优势卵泡+不注射FSH、去优势卵泡+注射FSH、去优势卵泡+不注射FSH处理供体牛,处理后第4天应用B型超声波导引活体采卵,试验重复10次。结果显示:注射FSH前去优势卵泡对其可采卵泡数、回收卵母细胞数和可用卵母细胞数均有显著提高(P0.05),但不注射FSH时去优势卵泡对提高采卵效率无显著效果;活体采卵对供体牛卵巢虽造成一定损伤,但停止采卵2~3个月后均能恢复正常发情、配种后能正常妊娠,对供体牛繁殖性能无显著影响。     

Effect of suckling on embryo production by repeated ovum pick-up before and after timed artificial insemination in early postpartum Japanese black cows

We investigated whether suckling would affect embryo production of cows bred by timed artificial insemination (TAI) following an ovulation synchronization protocol combined with ovum pick-up and progesterone releasing intravaginal device (OPU-PRID-TAI protocol). The number of oocytes and transferable embryos collected by repeated OPU, performed before and after TAI, were recorded. A total of 14 Japanese Black cows were divided into weaned (n=7) and suckled groups (n=7). All 14 cows were treated with OPU on day 0 (the first day of treatment) and then with a PRID for 9 days. Prostaglandin F(2alpha) analog was administered on day 7, GnRH analog was administered on day 10 (36 h after removal of the PRID) and TAI was performed 12 h later. Ovulation was confirmed by palpation per rectum the following day. After TAI, additional OPU sessions were performed on days 18, 25 and 32. The synchronized ovulation rates of the weaned and suckled groups were 100 and 85.7%, and the conception rates were 71.4 and 42.9%, respectively. Immature oocytes were fertilized and cultured in vitro. The numbers of oocytes collected and blastocysts generated were similar between the individual OPU sessions in both groups. However, the total numbers of oocytes collected, cultured oocytes, cleavage embryos and blastocysts as well as the proportions of cleavage embryos and blastocysts to cultured oocytes were all significantly (P<0.05) greater in the weaned group compared with the suckled group. These results suggest that the OPU-PRID-TAI protocol has the potential to produce a significant number of good-quality embryos in vitro after repeated OPU in early postpartum weaned Japanese Black cows. To collect more oocytes and produce more embryos, we suggest that calves be removed from cows scheduled for treatment using this protocol.     

Effect of Taurine and Melatonin in the Culture Medium on Buffalo In Vitro Embryo Development

This study was carried out to investigate the effect of supplementing culture medium with different concentrations of taurine and melatonin, on buffalo oocyte in vitro meiotic maturation and embryo development. In experiment 1, oocytes were matured in vitro and the cleaved embryos were cultured in the same following seven culture medium; (i) control (TCM 199 + 10% SS); (ii) control + 0.5 m m taurine; (iii) control + 1 m m taurine; (iv) control + 3 m m taurine; (v) control + 5 μ m melatonin; (vi) control + 10 μ m melatonin and (vii) control + 50 μ m melatonin. In experiment 2, based on the results of experiment 1, to examine the synergistic effect of antioxidants, the oocytes were matured in culture medium (TCM199 + 10% SS), supplemented with both taurine at 1 m m and melatonin at 10 μ m concentration and the cleaved embryos were cultured in the same medium. Supplementation of taurine at 1 m m concentration in the culture medium resulted in a higher (p < 0.05) transferable embryo (TE) yield when compared with control (20.6% vs 14.1%). Supplementation of melatonin at 10 and 50 μ m concentration in the culture medium resulted in a higher (p < 0.05) meiotic maturation rate (90.3% and 88.8% respectively) and TE yield (28.4% and 27.2% respectively), than the other treatments. In experiment 2, the TE yield did not improve by supplementing the culture medium with both taurine and melatonin, when compared with melatonin alone. In conclusion, the results of this study demonstrated that, enriching the culture medium with taurine and melatonin, improves in vitro embryo production efficiency in buffaloes. In particular, a high TE yield was obtained by enriching the culture medium with 10 μ m melatonin.     

Ovum pick-up and in vitro maturation in spotted paca (Cuniculus paca—Linnaeus, 1766)

We tested FSHp, eCG and FSHp + eCG to establish ovum pick-up (OPU) and in vitro maturation method in spotted paca. Eight healthy adult females were subjected to each of four treatments to stimulate ovarian follicular growth. All females were subjected to a hormonal protocol using a single dose of 45 mg of injectable progesterone and single intramuscular injection of 0.075 mg d-cloprostenol on day 6. Ovarian stimulation was carried out as follows: in Group TFE (FSHp and eCG), animals were treated with a single dose of 80 mg of FSHp and 200 IU of eCG intramuscularly on day 6 after the application of progesterone; in Group TF (FSHp), they were treated with a single dose of 80 mg of FSHp intramuscularly on day 6 after application of progesterone; in Group treatment eCG, they were treated with 200 IU of eCG intramuscularly on day 6 after application of progesterone; and in Group TC (saline solution), 1 ml of saline solution was administered to control does. The OPU was performed between 22 and 26 hr after gonadotropin treatments. All recovered oocytes were placed into maturation media and incubated for 24 hr. There were no differences among the mean number of observed follicles, aspirated follicles and oocytes recovered per treatment. Oocyte maturation rates did not differ among groups, except, TF and treatment eCG oocytes had greater maturation rates than TC oocytes. In this study, gonadotropin administration failed to superovulate treated does and increase oocyte retrieval efficiency. Despite the feasibility of the procedure, further studies are needed to develop and refine hormonal protocols for oocyte recovery and in vitro maturation in this species.     

牛活体采卵技术研究进展

活体采卵(Ovum Pick-up,OPU)可生产大量遗传品质优良、系谱明确的体外受精胚胎,成为牛胚胎移植的重要胚胎来源,在奶牛和肉牛生产中得到广泛应用。本文综述了牛OPU技术特点和优势,重点分析了影响OUP效率的因素,以期为牛OUP胚胎生产提供参考。