Quality and Fertilizing Ability In Vivo of Sex‐Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex‐sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX^® flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14‐propidium iodide), mitochondrial function (JC‐1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 × 10⁶ X‐bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non‐sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post‐thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.     

Fertility in Dairy Cows After Artificial Insemination Using Sex‐Sorted Sperm or Conventional Semen

The aim of this study was to compare pregnancy per artificial insemination (P/AI) after timed AI with sex‐sorted sperm (SS) or conventional semen (CS) in lactating dairy cows. Cyclic cows (n = 302) were synchronized by Ovsynch and randomly assigned into two groups at the time of AI. Cows with a follicle size between 12 and 18 mm and clear vaginal discharge at the time of AI were inseminated with either frozen‐thawed SS (n = 148) or CS (n = 154) of the same bull. A shallow uterine insemination was performed into the uterine horn ipsilateral to the side of probable impending ovulation. Pregnancy per AI on Day 31 tended (p = 0.09) to be less for SS (31.8%) than CS (40.9%). Similarly, P/AI on Day 62 was less (p = 0.01) for cows inseminated with SS (25.7%) compared with CS (39.0%). The increased difference in fertility between treatments from Days 31 to 62 was caused by the greater (p = 0.02) pregnancy loss for cows receiving SS (19.2%) than CS (4.8%). Cow parity (p = 0.02) and season (p < 0.01) when AI was performed were additional factors affecting fertility. Primiparous cows had greater P/AI than multiparous cows both on Day 31 (41.7% vs 25.0% in SS and 53.0% vs 31.8% in CS groups) and on Day 62 (33.3% vs 20.5% in SS and 48.5% vs 31.8% in CS groups). During the hot season of the year, P/AI on Day 31 was reduced (p = 0.01) in the SS group (19.6%) when compared with the rates during the cool season (38.1%). In conclusion, sex‐sorted sperm produced lower fertility results compared to conventional semen even after using some selection criteria to select most fertile cows.     

Effect of α-Tocopherol on Bovine In Vitro Fertilization

The objectives of this work are to determine if exogenous supplementation with α-tocopherol increases the in vitro fertilization (IVF) rate of bovine oocytes and improves viability of selected spermatozoa after 'swim-up'. The percentage of fertilized oocytes was significantly but negatively correlated ( r  = −0.941, p < 0.01) with the concentration of α-tocopherol. The control resulted in 95% of fertilized oocytes, which decreased as follows: 25 μM α-tocopherol (α25) 86%, 50 μM α-tocopherol (α50) 74%, 100 μM α-tocopherol (α100) 66% and 200 μM α-tocopherol (α200) 56%. Relatively to sperm viability after 'swim-up' with α-tocopherol supplementation, this antioxidant proved to have a beneficial effect as its concentration increased up to α50, decreasing for the concentrations of α100 and α200. Control resulted in 83% of live cells and 16% of dead cells; α25 resulted in 88% of live cells and 12% of dead cells; α50 resulted in 91% of live cells and 9% of dead cells; α100 resulted in 67% of live cells and 33% of dead cells; and finally α200 resulted in 57% of live cells and 42% of dead cells. In summary, the present study allows to conclude that, in our conditions, supplementation with the antioxidant α-tocopherol in IVF of bovine oocytes has a detrimental effect on fertilization rates. Nevertheless, exogenous supplementation with α-tocopherol at a concentration of 50 mM in the sperm-TALP media during the 'swim-up' technique has a significant beneficial effect on the selected spermatozoa viability.     

两种不同方法处理牛分离精子对体外受精效果的影响

本试验旨在探讨采用2种不同方法处理牛分离精子对精子活力、浓度及体外受精（IVF）效果的影响。将体外培养成熟卵母细胞随机分为2组,与分别用洗涤法和Percoll法处理牛分离精子进行IVF,并观察处理后各组的精子活率、浓度和IVF后囊胚发育率。结果显示,洗涤法和Percoll法处理后精子活力和IVF后的囊胚发育率均无显著差异,但采用洗涤法处理后分离精子浓度极显著高于Percoll法,可显著提高分离精子利用效率,且方法简便、经济,应优先选择使用。     

Rescue In Vitro Fertilization Method for Legacy Stock of Frozen Mouse Sperm

Sperm cryopreservation has been widely adopted for maintenance of the genetically engineered mouse (GEM). The cryopreserved sperm are being exchanged among many institutes worldwide. However, the recipients are not always able to obtain high fertilization rates with the frozen sperm shipped from senders. In this study, we cryopreserved mouse sperm via various methods and performed in vitro fertilization (IVF) in which the combination of methyl-beta-cyclodextrin for sperm preincubation and reduced glutathione for insemination was used (the MBCD-GSH IVF). In addition, frozen sperm sent from the Jackson Laboratory (USA) were thawed and used for IVF in the same manner. The fertilization rates of both the sperm cryopreserved via the methods applied in some countries and the cryopreserved GEM sperm improved when used with the MBCD-GSH IVF method. Therefore, we strongly believe that the MBCD-GSH IVF method brings about relatively high fertilization rates with any strain of frozen mouse sperm.     

牛体外受精的精子获能方法对受精率和胚胎发育的影响

从屠宰场获得牛卵巢,采取卵母细胞,成熟培养用ＴＣＭ１９９＋犊牛血清;精子获能处理,以ＢＯ液＋肝素钠为基础,三个试验组分别添加咖啡因、蔡碱和已酮可可碱,受精经分别为８１．３％、８３．３％、８８．９％,均显著高于对照组（Ｐ〈０．０１）,三个试验组的囊胚率分别为２０．４％、１９．３％、２１．２％。试验得出以下结论：囊胚率与受经成正比,添加已酮可要碱效果最好。另外组内种公牛成绩也有差异。     

影响精子受精力的因素

在人工授精及体外受精工作中，需要对精液进行稀释和处理，稀释及处理方法是否恰当，会直接影响精液的质量和受精能力。同时，稀释液中所添加的成分是否合适也会影响精液的质量和受精力。本文讨论精子处理过程以及稀释液中添加的有机物、无机离子、激素、维生素和细胞因子等对精子受精力的影响。     

精子不同处理方法对牛卵泡卵母细胞体外受精的影响

本研究探讨了不同精子处理方法对南阳黄牛卵泡卵母细胞体外受精效果的影响。试验一,研究三种不同的精子洗涤方法对卵母细胞体外受精的影响,结果表明:上浮法和Percoll密度梯度离心法所得的卵裂率(58.3%,60.6%)均显著高于直接离心洗涤法(47.2%,P<0.05);上浮法所得囊胚发育率(25.9%)显著高于直接离心法(13.3%,P<0.05);上浮法和Percoll法相比,卵裂率和囊胚率均无显著差异。试验二,研究三种受精液对卵母细胞体外受精的影响,结果表明:BO液组和mTyrode’s液组所得的卵裂率(60.1%,59.3%)均显著高于TCM199液组(48.5%,P<0.05);但三组囊胚率(17.8%、19.6%、14.1%)差异不显著(P>0.05)。     

牛体外受精胚胎的体外培养研究进展

家畜体外受精（IVF）技术自上世纪八十年代获得成功以来，发展十分迅速，九十年代已进入试管胚的开发应用阶段。目前，IVF胚胎的质量与体内胚相比仍有很大差距。在进行IVF胚胎体外培养时，常会出现“体外发育阻断”现象，主要表现为细胞数较少、卵裂球形状不规则、存在死亡的细胞和细胞质碎片、发育速率和抗冻性降低以及酶活性和营养物摄取的改变等方面。近年来，为了克服胚胎体外发育所存在的某些缺陷，国内、外对不同动物的胚胎体外培养体系和培养方法进行了系统研究，取得了长足进展。本文就牛IVF胚胎体外培养方面的研究进展综述如下。     

不同因素对奶牛分离精子ICSI效率的影响

本研究旨在探讨不同的联合激活处理、精子预处理方法和单精子注射方式对奶牛分离精子ICSI效率的影响。借助显微操作仪将经不同预处理的奶牛分离X精子直接注入体外成熟22～24h的牛卵母细胞胞质内,注射过程采用回吸胞质和不回吸胞质2种处理,最后分别用3种激活方案对注射卵进行激活。结果表明:用CR1aa+Ionomycin+6-DMAP对ICSI注射卵进行激活后,卵裂率和囊胚率都高于A23187+CHX和7%ET+CHX两种联合激活组(63.74%vs61.09%、53.75%;28.54%vs17.68%、22.00%,P0.05);采用percoll密度梯度离心法处理精子,卵裂率(77.10%vs62.19%,P0.05)与囊胚率(26.95%vs22.82%,P0.05)均高于上游法处理组;ICSI时回吸胞质获得的注射卵,其卵裂率和囊胚率均显著高于不回吸胞质处理组(63.10%,25.35%vs41.56%,19.40%,P0.05)。结果表明,用percoll密度梯度离心法处理精子、注射时回吸胞质、用CR1aa+Iono-mycin+6-DMAP激活ICSI注射卵,可显著提高奶牛分离精子ICSI的效率。     

Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.     

Effects of Addition of Tissue‐Type Plasminogen Activator in In Vitro Fertilization Medium on Bovine Embryo Development and Quality

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue‐type plasminogen activator (t‐PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus‐oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t‐PA and/or its inhibitor epsilon‐aminocaproic acid (control, t‐PA, t‐PA+ε‐ACA, ε‐ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t‐PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε‐ACA. PAA in the treated group was significantly reduced by ε‐ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t‐PA‐modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t‐PA. In conclusion, it appears that excessive t‐PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.     

Birth of Kids After Artificial Insemination with Sex‐Sorted,Frozen‐Thawed Goat Spermatozoa

Successful sex‐sorting of goat spermatozoa and subsequent birth of pre‐sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm‐sorting (using a modified flow cytometer, MoFlo SX^®) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post‐sorting and (ii) frozen in Tris–citrate–glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled‐rate freezer. Post‐sort and post‐thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC‐PNA). Sex‐sorted goat spermatozoa frozen in pellets displayed significantly higher post‐thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex‐sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex‐sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non‐sorted goat spermatozoa, non‐sorted ram spermatozoa and sex‐sorted ram spermatozoa. Following intrauterine artificial insemination with sex‐sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non‐sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex‐sorted by flow cytometry, successfully frozen and used to produce pre‐sexed kids.     

In Vitro Development of Bovine Embryos after Fertilization using Semen from Different Donors

Contents: The aim of this study was to determine whether the semen donor and/or heparin concentration influences the rate of fertilization of bovine follicular oocytes and their subsequent embryonic development in vitro. Frozen-thawed semen from five highly fertile bulls was treated with one of four concentrations of heparin (0.5,1.0, 2.0 and 5.0 μg/ml) on a 5 x4 factorial basis in an IVM-NF programme. Zygotes/oocytes were cultured in frozen-thawed bovine oviduct cell-conditioned medium for 6 days. The use of semen from different bulls resulted in significantly (P < 0.001) different rates offertilization, as judged by cleavage rates of the oocytes at 72 h post insemination, and subsequent embryonic development through the'8-cell-block'(P < 0.05) in vitro. Development up to the morula/blastocyst stage, however, did not differ significantly (P = 0.06) among groups of oocytes fertilized with spermatozoa from different bulls. Heparin levels ranging from 0.5 to 5.0 μg/ml did not differ in their effect on in vitro fertilization as judged by the rate of normally cleaved oocytes (P = 0.14). The overall parthenogenetic division rate at 72 h post insemination was 12.4% and was not influenced by the heparin concentration. There was a linear relationship (P < 0.001) between fertility estimates based on AI and the estimates basedon the first cleavage following in vitro fertilization.     

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 μ m β-mercaptoethanol (β-ME) and 50 μ m cysteamine (Cyst)] or a pro-oxidant (5 m m buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for β-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.     

The Effect of Oxidative Stress on Thawed Bulk‐Sorted Red Deer Sperm

The aims of this study were to assess the effects of the sex‐sorting process on post‐thaw sperm quality as well as on induced oxidative stress damage (H₂O₂ 0 mm  = H000; H₂O₂ 50 mm  = H050; H₂O₂ 100 mm  = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non‐sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H₂O₂ increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm‐sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non‐sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.