Application of platelet-rich plasma in the in vitro production of bovine embryos

Tropical Animal Health and Production - The aim of this study was to replace fetal bovine serum (FBS) with platelet-rich plasma (PRP) for in vitro production of bovine embryos. The maturation media...     

牛胚胎体外生产与早期胚胎发育的探讨

试验对牛胚胎体外生产技术中的体外受精液、精卵共育时间、血清和培乔液等影响早期胚胎体外发育的因素进行了研究。以BO（Brackett ＆ Oliphant）和BM（BO：成熟培养液=3：2）作为受精液，受精卵的囊胚发育率分别为26．0％和15．0％；精卵共育时间以9-12h为宜；受精卵在含血清FBS或OCS培养液中的专胚发育率分别为26．4％或29．9％，明显高于在无血清培养基中的囊胚发育率（10．3％）；TCMl99、mBECMaa、mSOFaa3种胚胎培养液均能支持受精卵的体外发育，在其中培养受精卵囊胚发育率分别为18．O％、30．7％和29．2％。试验结果表明：精卵于BM受精液中共育9～12h后，将假定受精卵置于添加5％OCS的mBECMaa或mSOFaa培养液中培养，能显著提高体外生产胚胎的囊胚发育率。     

Culture system and long-term storage of culture media in the in vitro production of bovine embryos

The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed.     

牛卵泡液对牛卵母细胞体外成熟及受精胚发育力的影响

研究了牛卵母细胞体外成熟液和胚胎培养液中添加不同浓度的牛卵泡液对其体外成熟率和受精胚发育力的影响。结果表明:添加10%牛卵泡液的实验组,卵母细胞的成熟率与血清对照组没有显著差异(P>0.05);添加10%卵泡液的实验组与血清对照组相比,卵裂率和囊胚率没有显著差异,却显著高于添加5%和20%牛卵泡液的实验组(P<0.01),且各实验组囊胚内细胞数差异不大(P>0.05)。因此,用10%的牛卵泡液可以取代成熟液和胚胎培养液中的血清,并可降低实验成本。     

牛体细胞核移植胚胎的批量化生产

利用2枚显微去核后的半卵与1枚供体细胞同步融合,并结合微穴法(WOWs)培养体系批量化生产成年牛克隆胚胎(日产40～80枚).结果,重构胚电融合率95.7%(3059/3197)、卵裂率87.1%(2637/3027)、囊胚率41.1%(1244/3027)和可冻胚率(72.5%,933/1244)均达到较高水平.克隆胚胎采用玻璃微管玻璃化冷冻保存数月后移植,30日龄时妊娠率为28.1%(48/171),已经产下5头足月克隆牛续.结果表明,该方法具有产业开发潜力.     

Comparison of persistence of seven bovine viruses on bovine embryos following in vitro exposure

The ability of seven cytopathic strains of bovine viruses to adhere to the zona pellucida of six-to-eight day-old bovine embryos were compared. Embryos were exposed to virus by placing them either in virus suspensions or by culturing them on infected bovine turbinate cultures for 18-24 h. After exposure to bovine virus diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBV), bluetongue virus (BTV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), parainfluenza 3 virus (PI3), or bovine enterovirus virus (BEV), the embryos were tested for virus by culture in bovine turbinate cells and by morphological examination using electron microscopy (EM). A special technique to minimize loss of embryos processed for EM was developed. More embryos had viral particles on the surface of the zona pellucida after exposure to 18-24 hour infected cell cultures than did embryos exposed to viral culture suspensions. The most dramatic finding was that BTV adhered in large numbers to the surface of the zona pellucida of exposed embryos. IBRV, PRV, and VSV comprised an intermediate group, with virions occasionally detected on the surface of exposed embryos after 5 washes. Therefore, extensive washing is required. The PI3 and BEV were easily removed from embryo-exposed virus by washing. BVD was difficult to identify morphologically, making assessment by EM unreliable. There was no evidence that any one of the seven viruses penetrated the intact zona pellucida. Using a micromanipulator, 42 embryos were also directly inoculated through the zona pellucida with +/- 50 picoliters of virus inoculum or medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in calf production by the transfer of bisected bovine embryos

A total of 132 embryos were recovered from 17 superovulated donor cows 7 d after estrus. Seventy-four embryos were selected and assigned to 2 treatment groups. The number of whole embryos that were directly transferred (Group A) and bisected (Group B) were 44 and 30 embryos, respectively. Sixty demi-embryos were produced from 30 morulae to blastocyst-stage embryos that were bisected. One hundred-three embryos, including whole and demi-embryos without zonae pellucidae, were nonsurgically transferred. Only one whole or demi-embryo was transferred to each recipients. The pregnancy rate for whole embryos (A) was 63.6% (28/44), while for demi-embryos (B) it was 74.6% (44/59). There was no significant difference between the pregnancy rates of whole embryos (A) and bisected embryos (B) transferred 7 d after estrus. Forty-three calves including the 14 sets of identical twins were obtained from 30 original embryos (143.3%) using the embryo bisection technique.     

Effect of phosphonoformic acid in the development of bovine embryos in vitro

This research evaluated the ability of phosphonoformic acid to inhibit bovine herpesvirus 1 (BHV-1) in cumulus cells commonly used in co-culture with bovine in vitro-produced embryos. At 200 and 400 microg/ml, phosphonoformic acid inhibited 4 logs of BHV-1. Subsequently, phosphonoformic acid (200 and 400 microg/ml) added to both in vitro fertilization and culture medium resulted in a decrease in the proportion of developed blastocysts, and the number of cells per blastocyst was lower in the treated embryos. Therefore, while phosphonoformic acid can effectively inhibit replication of BHV-1 in co-culture cells, it also inhibits development of in vitro-produced bovine embryos.     

In vitro development of bovine embryos encapsulated in sodium alginate

The objective of this study was to evaluate the in vitro development of bovine embryos encapsulated in alginate. Day-4 embryos produced in vitro (n = 110) were encapsulated with 1.5% sodium alginate and co-cultured with oviduct cells. Unencapsulated embryos (n = 106) were used as controls. In vitro development rate to the blastocyst stage at Day 7 was similar between encapsulated, 42.7%, (47/110) and control. 34% (36/106). embryos. Although encapsulated embryos were able to hatch on Day 9, they did so in a lower proportion than controls (P < 0.05). In conclusion, alginate encapsulation of bovine embryos does not disturb the in vitro development up to the blastocyst stage but significantly reduces the hatching process.     

In vitro interferon production by bovine tissues: effects of hydrocortisone.

The effect of hydrocortisone on bovine interferon production in vitro was studied. Infectious bovine rhinotracheitis virus was used as an inducer. Interferon was assayed by the plaque-reduction method in bovine fetal kidney cultures, using vesicular stomatitis virus as challenge virus. Hydrocortisone decreased interferon production in bovine fetal spleen and peripheral blood leukocyte cultures. Hydrocortisone did not decrease interferon production by bovine alveolar macrophages, in 1 experiment. Properties of viral inhibitors were those of interferon.     

Transition of cell numbers in bovine preimplantation embryos: in vivo collected and in vitro produced embryos

The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0.16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos.     

Effect in vitro of bovine viral diarrhea virus on bovine embryos with the zona pellucida intact,damaged and removed

The in vitro effect of bovine viral diarrhea virus (BVDV) on the survival of day 7 to day 7.5 bovine embryos collected from superovulated donors was studied. Fifty-four experimental embryos with the zona pellucida (ZP) intact, damaged or removed were exposed to 1×10⁴ TCD₅₀/ml of the NADL cytopathic strain of BVDC at 37°C for 24 hrs and compared to 36 control embryos that were cultured for 24 hr. Seven embryos with the ZP-removed were similarly exposed for 48 hrs and compared to five control embryos. The overall survival rate was 68% for embryos exposed to BVDV for 24 hrs and 77% for embryos not exposed (P>0.05). Extended exposure of the embryos with the ZP removed to virus for 48 hrs did not affect their survival rate compared to controls. Damage to the ZP by cracking or total removal of the ZP by micromanipulation or acidic Tyrode's solution had no effect on subsequent embryonic survival in the presence of BVDV. It was concluded that exposure to BVDV in vitro is not cytopathic for morula and blastocyst stage bovine embryos over a 48 hr period, even when they are not protected by the ZP.     

Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus–oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro‐fertilized or activated with ionomycin and cultured in vitro for 192 hr post‐in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat‐shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down‐regulated the expression of AQP3 (p < .01) and up‐regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down‐regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat‐shocked oocytes.     

Seasonality and photoperiod influence in vitro production of buffalo embryos

Buffalo is considered short-day breeder in tropical and subtropical part of the world and seasonality and photoperiodism impart major influence on its fertility. However, its impact on in vitro embryo production (IVEP) remains elusive. Therefore, this study investigated the effect of seasonal variations and photoperiodism on morphological and molecular parameters of IVEP in buffalo. For this purpose, we conducted two different experiments on the oocytes obtained by aspirating follicles from abattoir derived ovaries. In Exp. I, retrospective analysis was performed for oocyte recovery, blastocyst and hatching rate, during four consecutive seasonal periods (i.e. January–March, April–June, July–September and October–December). In Exp. II, oocytes from peak breeding and non-breeding seasons were subjected to 24 hr in vitro maturation and evaluated for polar body extrusion to assess maturation rate. Results showed that embryo development was markedly low during second quarter (April–June) and maximum during fourth quarter (October–December) of the year; referred as non-breeding and breeding seasons, respectively. Comparative data analysis demonstrated that poor oocyte quality is major reason for lesser efficiency of embryo production during non-breeding season than peak breeding season as suggested by poor oocyte recovery (2.31 ± 0.10 vs. 3.65 ± 0.27) and maturation rate (33.32 ± 2.1 vs. 63.15 ± 7.31). Subsequently, comparative gene expression analysis of blastocysts during peak breeding season significantly upregulated pluripotency gene (OCT-4) and downregulated heat shock protein 90, as compared to non-breeding season. Therefore, it could be divulged from the present study that seasonal variations and photoperiodism have profound effect on oocyte quality and subsequent embryo development. It is recommended to find suitable additives for in vitro maturation that could mitigate seasonal effects.     

2细胞期胚胎采集时间对电融合方法体外制备牛四倍体胚胎效率的影响

通过检测牛早期胚胎分裂速率的时间分布,并将体外受精后0~34 h与26~30 h时间段内采集的2细胞期胚胎分别作为电融合对照组与电融合试验组。将对照组与试验组的2细胞期胚胎分别用于电融合,并将电融合后的胚胎继续体外培养至囊胚期,检测并对比分析2组胚胎的融合率、分裂率、囊胚率和四倍体制备率。结果显示,牛2细胞期胚胎在体外受精后28~30 h其内分裂速率达到最高峰(18.9%)。电融合对照组的融合率、分裂率略高于试验组胚胎(85.2%vs.84.2%,82.2%vs.80.9%),试验组的囊胚率略高于电融合对照组(45.4%vs.44.8%),但差异均不显著。而电融合试验组的四倍体制备率显著高于电融合对照组(28.3%vs.14.5%)。结果表明,牛2细胞期胚胎的采集时间对电融合方法体外制备牛四倍体胚胎的效率有显著影响。     

秋水仙胺对电融合方法体外制备牛四倍体胚胎的影响

通过添加秋水仙胺从而改善电融合方法体外制备牛四倍体胚胎的过程。首先检测牛早期胚胎的分裂时间,并由此确定于体外受精后26~30h时间段内挑选既同期化且优质的2细胞期胚胎用于电融合。电融合参数选取2次直流电压为0.75kV/Cm且脉冲时间60μs。并通过免疫荧光染色监测电融合后细胞核的重组过程。设立电融合对照组和电融合秋水仙胺处理组,处理组在电融合后随即处理0.05mg/L秋水仙胺6h,后彻底洗涤继续体外培养至囊胚期。处理组的融合率（84.4%vs 84.8%）、分裂率（74.3%vs 77.6%）和囊胚率（36.1%vs 39.4%）皆低于对照组,但差异均不显著。获取的囊胚期胚胎的核型分析结果显示,处理组的四倍体制备率显著高于对照组（59.4%vs 26.9%）。综上所述,对电融合后的胚胎处理0.05mg/L秋水仙胺6h显著提高了牛四倍体电融合方法体外制的备率。     

In vitro and in vivo effects of recombinant bovine interferon-tau on bovine leukemia virus

The antiviral effects of recombinant bovine interferon-tau (rboIFN-tau) on bovine leukemia virus (BLV) were examined in vitro and in vivo. In the in vitro experiments, BLV titers decreased in FLK-BLV cells and in peripheral blood mononuclear cells of BLV-infected cattle treated with rboIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the in vivo effects of rboIFN-tau, 10 BLV-infected cattle were subcutaneously injected with rboIFN-tau. In the first experiment, 6 cows were administrated with 10(5) U/kg body weight of rboIFN-tau 3 times per week for 4 weeks, while in the second experiment 4 cows were administrated with 10(6) U/kg body weight of rboIFN-tau 3 times per week for 3 weeks. No adverse effects were observed after the administration of rboIFN-tau. In experiment No. 1, the mean BLV titers in cattle decreased in the post-rboIFN-tau administration period compared to the pre-rboIFN-tau administration period. In experiment No. 2, the mean BLV titers in cattle decreased in the rboIFN-tau administration period. These results suggest that rboIFN-tau decreases BLV titers in vitro and in vivo and that rboIFN-tau possibly reduces the degree of BLV titer in cattle without severe side effects.