黄牛利用活体采卵的应用研究

应用活体采卵仪对两组黄牛进行活体采卵.对黄牛每周2次和每周1次进行活体采卵,平均获卵分别为5.30枚、4.75枚,两者比较差异不显著(P>0.05);回收率分别为64.92%和81.85%,每周1次采卵回收率显著高于每周2次(P<0.05);优质卵母细胞率分别为80.12%和65.79%(P<0.01).健康和非健康牛只采卵,平均获卵母细胞数分别为6.71枚、5.10枚(P<0.05);回收率分别为65.28%、62.20%,无明显差异(P>0.05);优质卵母细胞率分别为79.91%、73.37%,差异不显著(P>0.05).     

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.     

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.     

Improved embryo development in Japanese black cattle by in vitro fertilization using ovum pick-up plus intracytoplasmic sperm injection with dithiothreitol

The purpose of this study was to determine whether dithiothreitol (DTT) treatment of sperm and ethanol activation improve embryo production by intracytoplasmic sperm injection (ICSI). Further, we compared ICSI with standard in vitro fertilization (IVF) in oocytes obtained from cattle. We demonstrated that DTT reduced the disulfide bond in the bovine sperm head. Using oocytes obtained from a slaughterhouse, ICSI-DTT treatment without ethanol showed the highest rate of blastocyst formation. We applied these results to fertilization using ovum pick-up (OPU). Eleven Japanese black cattle served as donors for OPU plus standard IVF (OPU-IVF). Of them, four donors with low embryo development rates were selected to determine whether embryo development was enhanced by OPU plus ICSI (OPU-ICSI). We assessed effects on embryo development following IVF and ICSI in oocytes obtained using OPU. Blastocyst rates were significantly higher for OPU-ICSI than for OPU-IVF. Our results suggest that OPU-ICSI improves the blastocyst development rate in donors with low embryo production compared with the standard OPU-IVF.     

奶牛产后瘫痪综合防治技术

奶牛产后瘫痪是奶牛常见营养代谢性疾病。本文阐述了本病的临床症状、发病机理,介绍了某奶牛场对该病的综合防治技术方案。     

Accuracy and repeatability of clinical examinations postpartum in dairy cattle

A variety of diagnostic tests are utilized during herd health examinations at the herd and individual cow level. While most of these tests are based on the best available evidence it has to be considered that each diagnostic test generates type 1 and 2 errors. Such diagnostic errors can lead to treatment errors with relevant clinical and pharmacological as well as ethical and economical consequences. The objectives of our studies were to generate science-based information on the repeatability and accuracy of relevant diagnostic methods utilized in postpartum monitoring of dairy cows. Both rectal thermometry and visual examination of vaginal discharge by means of a speculum are not perfect. Results can be biased by a variety of factors. Diagnostic performance (accuracy and repeatability), however, is adequate for cow side tests in the practice. The visual assessment of rumen fill is an inadequate method to monitor dry matter intake in cows.     

The effects of twisting and type of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up in cattle

The effects of twisting and type (single- or double-lumen) of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up (US-guided OPU) were investigated in cattle. The first study using slaughterhouse ovaries revealed that twisting of the needle during follicle aspiration improved the oocyte recovery rate without deleterious effects on the attachment of cumulus layers. Vacuum pressure affected the oocyte recovery and cumulus attachment, regardless of the needle type. The needle type did not affect the oocyte recovery or cumulus attachment with an optimized vacuum pressure. In the second study, US-guided OPU was performed in live cows using two types of needles with a vacuum pressure of 75 mmHg. The needle type did not affect the oocyte recovery or cumulus attachment of the recovered oocytes. The results revealed that twisting of the needle is effective in follicle aspiration, and suggested that a single-lumen needle is as useful as a double-lumen needle for US-guided OPU in cattle.     

Energy balance and pulsatile LH secretion in early postpartum dairy cattle

The relationships between energy balance (EB), pulsatile LH secretion and circulating levels of insulin and various energy metabolites were assessed in ten lactating Holstein cows. Cows were blood sampled every 12 min via indwelling jugular catheters for 8 hr twice weekly beginning in the first week postpartum (PP) through first ovulation (1stOV). Days to negative EB nadir and days to 1stOV were highly correlated (P less than .02). LH pulse frequency (P less than .01), LH baseline (P less than .05) and mean LH (P less than .1) increased, while LH pulse amplitude tended to decrease when comparing the frequent sampling series immediately before and after the negative EB nadir. Plasma levels of non-esterified fatty acids (NEFA, r = -.40, P less than .01), insulin (r = .38, P less than .01), glucose (r = .31, P less than .01) and beta-hydroxybutyrate (r = .19, P less than .06) were correlated with EB. For within animal comparisons, NEFA was significantly correlated (r = -.44 to -.89, P less than .05) with EB in 8 of 10 cows, but no other correlations were significant. Additionally, none of the metabolites nor insulin were significant in multiple regression models for 1stOV. Therefore, it appears that pulsatile LH secretion is suppressed until the negative EB nadir is reached, at which time LH pulse frequency increases stimulating 1stOV. Since NEFA and EB are directly related, NEFA may serve as a peripheral signal of EB to the central nervous system.     

Association between postpartum pyrexia and uterine bacterial infection in dairy cattle

The temperature of 90 dairy cattle was recorded for the first 10 days after parturition and the animals were categorised as either normal (< 39.7 degreesC) or pyrexic. Swabs were collected from the uterine lumen seven, 14, 21 and 28 days after parturition for aerobic and anaerobic culture; bacteria were identified and their growth was scored semiquantitatively. Blood samples were collected three times a week for the estimation of the concentrations of acute phase proteins. The cows' temperatures were often above the accepted normal range, but it was not a good indicator of the number of bacteria in the uterus. However, pyrexia was correlated with the presence of specific uterine pathogens (P < 0.05) and in particular with Prevotella species (P < 0.01). The pyrexic animals had a higher plasma concentration of the acute phase protein (alpha1-acid glycoprotein (P < 0.05). Although pyrexia is an indicator of postpartum inflammation, additional clinical signs are necessary to identify uterine bacterial infection.     

Energy metabolites in pre- and postpartum dairy cattle as predictors of reproductive disorders

This study aimed to determine values for selected energy metabolites (non-esterified fatty acid [NEFA], β-hydroxybutyrate [BHB], urea or urea:BHB ratio), together with a body condition score, associated with an increased risk of cows developing a reproductive disorder and to investigate temporal relationships between predictors and reproductive outcome. A cohort of 98 cows on one farm was monitored weekly from four weeks before to 10 weeks after calving; 89 cows provided sufficient data to calculate commencement of luteal activity (C-LA). Cows with high NEFA × urea (Nu; product of NEFA and urea) values one and three weeks after calving were twice as likely to develop cystic ovarian disease (risk ratio 2). Cows that developed endometritis had high NEFA values one (P=0.02) or four weeks (P=0.04) before calving, or low urea:BHB ratios two weeks before calving, at calving or three weeks after calving (P=0.024, P=0.031 and P=0.001, respectively). Cows that had delayed C-LA had high NEFA values one week after calving (P=0.05) or low urea:BHB ratios three or four weeks after calving (P=0.004 and P=0.003, respectively).     

Alteration of the GnRH-induced LH release by steroids in postpartum dairy cattle

The effect of elevated plasma concentrations of estradiol-17 beta (E2 beta), estrone (E1) and progesterone (P), in concentrations similar to those observed at the end of pregnancy, on the gonadotropin releasing hormone (GnRH)-induced luteinizing hormone (LH) release in postpartum dairy cows was studied. Twenty-five dairy cows in late gestation were assigned to five groups of five each to receive daily steroid treatments as follows: 1 and 2) no exogenous steroids; 3) 20 mg E2 beta and 30 mg E1; 4) 150 mg P and 5) 20 mg E2 beta + 30 mg E1 + 150 mg P. Steroids were dissolved in alcohol (vehicle) and injected sc twice daily. Cows receiving no steroids were given vehicle. Administration of steroids or vehicle began immediately after parturition (d 0) and continued for 7 d to maintain concentrations of steroids in plasma similar to prepartum concentrations. Cows in groups 2 through 5 received an injection of 100 micrograms GnRH on d 2, 8, 16, 24 and 32 postpartum, while those in group 1 received water (vehicle for GnRH) on the same days. Plasma for hormonal determinations was collected on alternate days beginning 10 d before the expected day of parturition, daily through the period of steroid treatments (d 0 to 6, postpartum) and on alternate days thereafter until d 40 postpartum. In addition, plasma was collected immediately before GnRH or water administration and at .5 h intervals thereafter for 4 h. Trends in response to treatment over days postpartum were studied by partitioning sums of squares due to linear, quadratic and cubic polynomial responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicle culture enhances fertilizability and cleavage of bovine oocytes matured in vitro

The fertilization and cleavage of bovine oocytes matured by intra- or extra-follicular methods were investigated. Oocytes were fertilized in vitro or in the rabbit oviduct and cleavage was assessed after in vitro culture of in vitro fertilized oocytes and after in vivo culture (rabbit oviducts) of xenogenously fertilized oocytes. The effect of fertilization with fresh-diluted or frozen-thawed semen were also examined. The intra-follicular method did not increase the nuclear maturation rate as compared with the extra-follicular method (57.9 and 52.7%, respectively). However, the proportions of in vitro fertilized eggs (54.8%) and of cleaved eggs (two to eight cells; 34.6%) in the rabbit oviduct for 48 h after xenogenous fertilization were higher (P less than .025) in the intra-follicular oocytes than those of the extra-follicular oocytes (37.1 and 21.3%, respectively). It was also found that the use of fresh-diluted semen resulted in more cleaved eggs from the rabbit oviduct than the use of frozen-thawed semen (43.4 and 23.3% in the intra-follicular oocytes, P less than .025; 31.0 and 7.8% in the extra-follicular oocytes, P less than .05), while the appearance of cleaved eggs following in vitro fertilization was extremely low (0 to 6.6%). The present results demonstrated that the intra-follicular culture method of bovine oocytes provided a physiological environment for cytoplasmic maturation leading to higher fertilizability and development than the conventional in vitro culture of extra-follicular oocytes.     

聚乙烯吡咯酮与促卵泡素对奶牛活体采卵效率的影响

采用4×4拉丁方设计,研究聚乙烯吡咯酮(PVP)溶解促卵泡素(FSH)一次肌肉注射对奶牛活体采卵效率和体内血清FSH含量的影响。将8头荷斯坦空怀母奶牛随机分为4组,每组2头,分别用5mgFSH 5mL15%PVP(15%PVP组)、5mgFSH 5mL30%PVP(30%PVP组)、5mgFSH 5mL生理盐水(对照组)和常规注射FSH(常规组)处理供体奶牛。试验期4个月,每月处理1次,各组均为处理后4d应用B型超声波导引采卵。结果如下:30%PVP组每头次平均可采卵泡9个,获可用卵母细胞4枚,与常规组比较差异不显著(P>0.05),但与对照组比较差异显著(P<0.05);15%PVP组每头次平均可采卵泡6个,获可用卵母细胞2.25枚,与对照组比较差异显著(P<0.05);体内血清FSH浓度,30%PVP组与常规组变化趋势相似,维持适宜FSH浓度时间较长,15%PVP组次之,而对照组注射后4~12h血清FSH浓度较高,以后急剧下降,表明PVP可延长FSH在体内存留时间,但以30%PVP效果最佳。     

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。     

Development of vitrified matured cattle oocytes after thawing and culture in vitro

Bovine oocytes were partly denuded either at the beginning (t0) or six hours (t6) after the beginning of maturation and vitrified by the open pulled straw method at the end of the maturation process. After warming and fertilisation, their development in vitro and in vivo was assessed. The rates of production of blastocysts achieved in vitro were 3.4 per cent for the t0 group and 0.9 per cent for the t6 group compared with 40.4 per cent for the control oocytes. After transfer at the blastocyst stage pregnancies have been established in the three groups. Some of these pregnancies originated from vitrified oocytes which were further vitrified at the blastocyst stage before being transferred into synchronised recipients.     

Applicability of a progesterone-based timed artificilal insemination protocol after follicular fluid aspiration using the ovum pick-up technique in suckled beef cows

We conducted a progesterone-based timed AI protocol after follicular fluid aspiration using the ovum pick-up (OPU) technique to examine its applicability to the suckled beef cow. A total of 19 beef cows were randomly allocated to one of the following three groups based on the number of days postpartum: 13 to 60 days (Group A: suckled; early postpartum period, n=9), 61 to 150 days (Group B: suckled; mid postpartum period, n=6), or 151 to 281 days (Group C: non-suckled; prolonged open period, n=4) postpartum. These cows were treated with follicular fluid aspiration and insertion of a progesterone-releasing intravaginal device (PRID) on day 0. The PRID was removed and 500 microg of cloprostenol was intramuscularly administered on day 7. A dose (100 microg) of fertirelin acetate was injected intramuscularly 48 hours later, and this was followed by a timed AI (TAI) after another 18 hours (day 10). Serum samples were taken on days 0, 7, 9, 10, 12, 17, 24 and 31 for determination of the estradiol-17beta (E(2)) and progesterone concentrations. Pregnancy diagnosis was made by rectal palpation approximately 60 days after TAI. There was no significant difference in the peripheral E(2) concentrations among the three groups during the period of the hormonal treatment. The average progesterone concentrations in Group A on day 17 were significantly higher than those in Group B and exceeded 1.0 ng/ml on day 17 and thereafter. There was no significant difference in the numbers of collected immature oocytes among the three groups. The pregnancy rates in Groups A, B, and C were 77.8% (7/9), 83.3% (5/6) and 50.0% (2/4), respectively. In conclusion, this timed AI protocol is applicable to suckled beef cows within the period of 60 days postpartum.     

预防控制奶牛疫病确保乳业健康发展

乳制品已成为日常重要消费品，在乳制品相对充足条件下，消费者越来越重视其营养价值和安全性。欲求从根本上保证乳品安全，必须从源头即保障奶牛健康上下大气力；预防、控制奶牛疫病，才能确保乳制品的营养价值和安全性。