Age-Dependent Effect of Foal Heat Breeding on Pregnancy and Embryo Mortality Rates in Thoroughbred Mares

The aim of the present study was to evaluate the correlation of age and heat cycle to determine reproductive efficiency in young and aged Thoroughbred mares bred on foal heat (FH) or on second heat (SH) after foaling. Embryo mortality (EmbM) was determined every time a mare was found open after a positive pregnancy diagnosis. Parturition to breeding interval, pregnancy rate (PregR) and EmbM rate were the dependent variables and the treatments were breeding on the FH or on SH. The cutoff age to obtain above-average probability for the EmbM was 10 years old. PregR in mares bred on FH was lower compared with SH (P < .01); however, it was neither affected by the age of mares (P > .05) nor by the age group of mares (P > .05). Regarding FH and SH, there was a difference in PregR in young mares (P < .01), unlike in aged mares (P > .05). EmbM rate was not different between mares bred on FH or SH (P > .05) although it was affected by age of mares (P < .01). EmbM was higher in oldest than young mares (P < .01). Aged mares bred on FH had a significantly higher EmbM rate compared with the young group also bred on FH (P < .01). In conclusion, the reproductive efficiency of Thoroughbred mares bred on FH is dependent of the age. Aged mares (≥10 years old) should be bred at their SH to reduce EmbM and improve reproductive performance.     

Occurrence of Equine Coital Exanthema in Mares from an Embryo Transfer Center

Equine coital exanthema (ECE) is an acute, venereal disease caused by equid herpesvirus 3 (EHV-3), characterized by the formation of papules, vesicles, pustules, and ulcers on the vaginal and vestibular mucosa as well as on the skin of the penis, prepuce, and perineal region of mares and stallions. The present work describes an outbreak of ECE characterized by typical lesions around the anus and on the perineal skin, discomfort, and anorectal lymphadenopathy, which occurred in a large number of donor and recipient mares at an embryo transfer center in Argentina. We found that the horses' movement pattern, the environmental conditions, and the management procedures could have favored the introduction and/or reactivation from latency and spread of EHV-3. Although this is the first report of an outbreak of ECE in an embryo transfer facility, it reinforces the need to introduce additional hygienic and preventive measures to avoid EHV-3 spread and future ECE outbreaks.     

Advances in Holding and Cryopreservation of Equine Oocytes and Embryos

Methods for holding of oocytes and embryos during shipment as well as for their cryopreservation can greatly aid equine reproductive management. Oocytes can be held at room temperature overnight or at cooler temperatures for two nights without affecting maturation or embryo development after intracytoplasmic sperm injection. In contrast, methods for cryopreservation of equine oocytes that support high rates of embryo development have not yet been established. Equine embryos may be held overnight at temperatures from 5°C to 19°C without reduction in viability, but longer holding periods, or higher holding temperatures, may be detrimental. Small equine embryos (<300 μm), either in vivo derived or in vitro produced, can be slow frozen or vitrified successfully. In the last decade, methods have been developed to allow in vivo–derived expanded blastocysts, up to Day 8, to be vitrified successfully after blastocoele collapse. These methods of shipment and preservation allow mare owners in remote locations to have access to sophisticated assisted reproductive technologies.     

A History of Equine Embryo Transfer and Related Technologies

The first successful equine embryo transfer was reported in 1972, 21 years after the first reported embryo transfer in cattle. Adaptations of embryo transfer and the various related technologies have generally been more rapid in the equine than in cattle, with the exceptions of superovulation, in vitro fertilization and cryopreservation. Recent progress has been achieved in all three of these areas. This paper presents a time line of various events in the history of equine embryo transfer and related technologies. Approximately 25,000 equine embryo transfers are being conducted per year, worldwide.     

Factors Affecting Recipients' Pregnancy,Pregnancy Loss,and Foaling Rates in a Commercial Equine Embryo Transfer Program

During 11 breeding seasons, 351 7- to 10-day-old horse embryos were nonsurgically transferred into recipients that ovulated between 3 and 10 days earlier. Pregnancy rates at 14 and 40 days and foaling rates were 77.8% (273/351), 69.2% (243/351), and 64.4% (226/351), respectively. Pregnancy loss between 14 and 40 days was 11% and between 40 days and delivery was 7%. The transfer of quality grade 3 to 4 embryos resulted in a significantly lower pregnancy rate at 14 days compared with the transfer of grade 1 to 2 embryos (46.2% vs. 79%; P < .05). Eight-day-old embryos resulted in significantly lower pregnancy losses than day 9 or 10 embryos, as occurred for embryos between 400 and 1200 μm compared with embryos <400 μm. Embryos recovered from mares >20 years resulted in a significantly higher pregnancy loss rate than those recovered from younger mares. The same happened for embryos coming from mares affected by reproductive pathologies compared with healthy mares performing sport activity. None of the evaluated parameters influenced recipients' foaling rate significantly.     

鲜整胚及分割双半胚移植试验研究

本试验结合我省胚胎移植的现状,用7头纯种西门塔尔牛作供体,46头杂交牛作受体,进行同期发情和超数排卵,获得鲜整胚59枚,双半胚32枚;用鲜整胚22枚,双半胚24枚进行移植,研究鲜整胚与分割双半胚的移植效果。结果表明:鲜整胚单枚移植妊娠率为27.3%(6/22),分割双半胚移植妊娠率为54.2%(13/24),鲜整胚单枚移植妊娠率比分割双半胚移植妊娠率低26.9个百分点,经t检验差异显著(P<0.05)。     

Ultrastructural Morphology and Nuclear Maturation Rates of Immature Equine Oocytes Vitrified with Different Solutions and Exposure Times

Ultrastructural morphological injuries and maturation rates were investigated in equine oocytes exposed to vitrification solutions (VS) containing synthetic ice blockers (SIBs) during different exposure times. In experiment 1, compact cumulus-oocyte complexes (COCs; n = 30) were randomly allocated to treatments: (1) fresh fixed (control); (2) VS-1 (1.4 M dimethyl sulfoxide [DMSO] + 1.8 M ethylene glycol [EG] + 1% SIB) for 3 minutes of equilibrium time and VS-2 (2.8 M DMSO + 3.6 M EG + 0.6 M sucrose + 1% SIB) for 1 minute (Eq-long); and (3) VS-1 for 1.5 minutes and VS-2 for 30 seconds (Eq-short). In experiment 2, compact (n = 248) and expanded (n = 264) COCs were evenly distributed to the following treatments: (1) immediate maturation in vitro (control); (2) vitrification using the Eq-short protocol as in experiment 1; and (3) vitrification using a stock solution containing 2.8 M formamide, 2.8 M DMSO, 2.7 M EG, 7% polyvinylpyrrolidone, and 1% SIB (Eq-short-mod). More (P < .02) oocytes with normal ultrastructural morphology were seen in fresh control and Eq-short groups than in Eq-long group. Metaphase-II (MII) rates were higher (P < .05) for oocytes with expanded cumulus than compact cumulus in the control group, and higher (P < .05) for oocytes with expanded cumulus than compact cumulus in Eq-short and Eq-short-mod groups. No difference in MII rates was detected among groups within each type of COC. In conclusion, reduction of exposure time to VS better preserved oocyte ultrastructural features, and MII rates were higher for vitrified oocytes with expanded cumulus. This study advances our knowledge on potential alternatives for vitrification of immature equine oocytes.     

Effect of eFSH on Ovarian Cyclicity and Embryo Production of Mares in Spring Transitional Phase

The use of equine FSH (eFSH) for inducing follicular development and ovulation in transitional mares was evaluated. Twenty-seven mares, from 3 to 15 years of age, were examined during the months of August and September 2004, in Brazil. Ultrasound evaluations were performed during 2 weeks before the start of the experiment to confirm transitional characteristics (no follicles larger than 25 mm and no corpus luteum [CL] present). After this period, as the mares obtained a follicle of at least 25 mm, they were assigned to one of two groups: (1) control group, untreated; (2) treated with 12.5 mg eFSH, 2 times per day, until at least half of all follicles larger than 30 mm had reached 35 mm. Follicular activity of all mares was monitored. When most of the follicles from treated mares and a single follicle from control mares acquired a preovulatory size (≥35 mm), 2,500 IU human chorionic gonadotropin (hCG) was administered IV to induce ovulation. After hCG administration, the mares were inseminated with fresh semen every other day until ovulation. Ultrasound examinations continued until detection of the last ovulation, and embryo recovery was performed 7 to 8 days after ovulation. The mares of the treated group reached the first preovulatory follicle (4.1 ± 1.0 vs 14.9 ± 10.8 days) and ovulated before untreated mares (6.6 ± 1.2 vs 18.0 ± 11.1 days; P < .05). All mares were treated with prostaglandin F_2α (PGF_2α), on the day of embryo flushing. Three superovulated mares did not cycle immediately after PGF_2α treatment, and consequently had a longer interovulatory interval (22.4 vs 10.9 days, P < 0.05). The mean period of treatment was 4.79 ± 1.07 days and 85.71% of mares had multiple ovulations. The number of ovulations (5.6 vs 1.0) and embryos (2.0 vs 0.7) per mare were higher (P < 0.05) for treated mares than control mares. In conclusion, treatment with eFSH was effective in hastening the onset of the breeding season, inducing multiple ovulations, and increasing embryo production in transitional mares. This is the first report showing the use of FSH treatment to recover embryos from the first cycle of the year.     

牛胚胎冷冻与移植受胎技术研究

本文进行了牛胚胎冷冻与移植受胎技术研究。摸索出了提高牛冷冻胚胎成活率和移植受胎率的综合配套技术。牛冷冻胚胎解冻成活率达８４．４％（５７／１２２）。其中１９９６年移植受胎率达到５５．６％（２０／３６）。Ａ级胚胎达以７７．８％（２１／２７），并在我国首次获得中国荷斯坦牛冷冻胚胎的二分胚的同孵孪生牛犊。且达到４２．９％（３／７）移植受胎率。     

小鼠卵母细胞和胚胎不同冷冻方法的比较研究

探讨程序化冷冻与玻璃化冷冻对小鼠GV期卵母细胞及二细胞期胚胎的复苏率及其发育潜能的影响。通过小鼠的卵母细胞与早期胚胎的不同冷冻方法的比较,为后续阿旺绵羊的胚胎冷冻保存提供参考。采用程序化冷冻与玻璃化冷冻技术,分别冷冻小鼠GV期卵母细胞及二细胞期胚胎,复苏后培养,比较不同冷冻处理后的复苏率、成熟率与囊胚率。小鼠GV期卵母细胞程序化冷冻复苏率(48.00%±5.29%)显著低于玻璃化冷冻复苏率(65.00%±5.00%),有统计学差异(P=0.0147<0.05);而程序化冷冻后复苏卵母细胞的发育成熟率略高于玻璃化冷冻组,但无统计学意义。小鼠二细胞期胚胎程序化冷冻组复苏率(76.00%±2.00%)显著高于玻璃化冷冻组复苏率(70.00%±2.00%),有统计学差异(P=0.0213<0.05);冷冻后复苏胚胎发育的囊胚率程序化冷冻略低于玻璃化冷冻及对照组,但无统计学意义。     

Effects of Sperm Concentration and Straw Volume on Motion Characteristics and Plasma, Acrosomal, and Mitochondrial Membranes of Equine Cryopreserved Spermatozoa

The advantages of using cryopreserved semen in equine reproduction are well known. During cryopreservation, spermatozoa undergo many changes that lead to a decrease in fertility. There is no agreement on the ideal sperm dose and concentration to maximize fertility rates. Thus, the objectives of this experiment were to evaluate sperm motion by computer-assisted analysis (CASA), sperm membrane integrity and function with fluorescence probes of cryopreserved sperm at three concentrations: 100 (C100), 200 (C200) and 400 × 10⁶ sperm/mL (C400), and two straw volumes (0.50 and 0.25 mL). There was no interaction between sperm concentration and storage volume (P > .05). Sperm motion characteristics were influenced by concentration (C100 > C200 > C400; P < .05). Curvilinear velocity (VCL) in 0.25-mL straws had higher average values (P < .05). Membrane integrity and function were not changed by straw volume (P > .05). However, sperm concentration changed the percentage of cells with intact plasma membrane (C100 > C200 > C400; P < .05) and the percentage of cells with high mitochondrial membrane potential (C100 = C200; P > .05 and C400 < C100 and C200; P < .05). According to this experiment, the best freezing method was that involving 100 × 10⁶ sperm/mL, regardless of straw volume.     

供体细胞的不同处理方法对牛核移植重构胚发育能力的影响

为了研究供体细胞不同的处理方法对核移植重构胚的作用，比较了用于保种的冻存和新鲜的成纤维细胞做供体细胞、供体细胞不同的离心转数、供体细胞不同的血清饥饿时间及用本实验室冻存的成纤维细胞，解冻复苏后，不同传代次数对重构胚的影响。结果表明分别用冷冻保存的和新鲜的成纤维细胞作为核供体，所得重构胚卵裂率、囊胚率无显著差（P>0.05）；供体细胞离心800 r/min时所得重构胚效果较好，离心1500 r/min所得重构胚的卵裂率、囊胚率与其他3组相比较低；血清饥饿3～5 d组所得重构胚比其他3组好；用解冻复苏后再传2、5代的细胞进行核移植，所得重构胚的卵裂率、囊胚率无明显差异(P>0.05)，显著高于传8代的细胞所得重构胚。说明供体细胞的不同处理方法对核移植重构胚发育有很重要的影响。     

Effect of a Nonsurgical Embryo Transfer Procedure and/or Altrenogest Therapy on Endogenous Progesterone Concentration in Mares

Endogenous progesterone levels may decline after transcervical embryo transfer in some mares. Progestogen therapy is commonly used to support endogenous progesterone levels in embryo transfer recipient mares or those carrying their own pregnancy. The goal of this study was to determine the effects of the transcervical transfer procedure and/or altrenogest therapy on luteal function in mares. Mares were assigned to one of six treatment groups: group 1 (untreated control; n = 7 cycles), group 2 (sham transfer, no altrenogest; n = 8 cycles), group 3 (sham transfer plus altrenogest; n = 8 cycles), group 4 (pregnant, no altrenogest; n = 9 mares), group 5 (pregnant plus altrenogest; n = 9 mares), and group 6 (nonpregnant plus altrenogest; n = 10 cycles). Mares in groups 4-6 were bred and allowed an opportunity to carry their own pregnancy. Blood samples were collected for 22 days beginning on the day of ovulation. Sham embryo transfer (groups 2 and 3, combined) did not result in a decline in endogenous progesterone levels compared with control mares (group 6). However, sham embryo transfer did result in luteolysis and an abrupt decline in endogenous progesterone levels in one of the 16 (6.2%) sham-transferred mares. Altrenogest therapy in sham-transferred mares (group 3) was associated with lower endogenous progesterone levels on days 10, 12, and 13 postovulation when compared with sham-transferred mares that did not receive altrenogest (group 2). Administration of altrenogest to pregnant mares (group 5) was associated with lower concentrations of endogenous progesterone from days 14 to 18 and on day 21 compared with endogenous progesterone levels in pregnant mares not administered altrenogest (group 4). In conclusion, a transcervical embryo transfer procedure can cause luteolysis in a low percentage of mares. Altrenogest therapy may be associated with a reduction in endogenous progesterone secretion, presumably mediated by a reduction in pituitary luteinizing hormone (LH) release and a decrease in luteotropic support.     

白绒山羊性控胚胎生产及移植应用研究初探

应用X性控冷冻精液对超排供体白绒山羊进行人工授精,生产性控胚胎并进行鲜胚移植。结果发现,①用性控冻精和鲜精输精的供体母羊,平均卵子回收数分别为13.3和12.5枚,其中性控冻精组受精卵比例为29.6%（55/186）,极显著低于鲜精组94.0%（281/299）的比例（P<0.01）;②性控胚胎和普通胚胎移植受体母羊产羔率分别为42.2% 和58.1% ,二者差异极显著（P<0.01）;③性控胚胎移植受体母羊所产羔羊雌性所占百分数为100%,极显著高于普通胚胎移植（P<0.01）。表明通过白绒山羊X、Y精子分离、人工授精生产性控胚胎,同时结合胚胎移植技术,可以达到按照生产实际中的意愿得到性别控制子代白绒山羊的目的。     

Influence of Steroidal Anti-Inflammatory Drugs on Viability and Fertility of Equine Semen

Considering the efficiency of steroidal anti-inflammatory drugs (SAIDs) in the immunomodulation of breeding-induced endometritis and the possibility of using these drugs by intrauterine route instead of the parenteral application, the present study aimed at evaluating the effect of SAIDs added to the semen extender on equine sperm viability and fertility. In experiment 1, 15 SAIDs were individually added to a skim milk-based extender and, based on the results of sperm motility, dexamethasone was the drug of choice for the subsequent trials. The effect of dexamethasone on the viability of fresh and 24-hour cooled semen was investigated in experiment 2. In experiment 3, fertility rate was measured in both post-breeding endometritis-resistant and susceptible mares. Although dexamethasone supplementation caused a premature decrease in sperm total and progressive motility and in sperm velocities (P < .05), no difference was observed for sperm membrane integrities and fertility (P > .05). Based on these results, we can conclude that dexamethasone can be added to equine semen at the time of insemination or before cooling, although its use was not able to increase fertility.     

不同电融合条件对莎能奶山羊转基因(GFP)核移植胚胎发育的影响

以莎能奶山羊（Saanen dairy goat）转基因（GFP）成纤维细胞为核供体细胞，以普通山羊卵母细胞为受体细胞进行了核移植，试验中对转基因核移植胚胎的电融合条件进行了优化，发现对于莎能奶山羊转基因（GFP）重构胚胎的处理，电融合时方案B（1．5kV／cm、3μs／次、间隔1s）电融合参数最适合用于莎能奶羊的转基因（GFP）核移植胚胎，其重构卵电融合率为89．4％，重构胚分裂率为68．0%。     

黄芩苷、川芎嗪对小鼠体外培养胚胎冷冻-解冻及移植效果的研究

将中药有效成分黄芩苷(Baicalin,Bai)和川芎嗪(Ligustrazine,Lig)添加到小鼠2-细胞胚胎培养液中进行体外培养,比较其体外发育能力,并将体外发育的桑椹胚、囊胚进行2种程序常规冷冻.解冻后形态正常的桑椹胚、囊胚分别培养8～14 h、6～8 h,比较各组胚胎的冷冻-解冻发育效果及冷冻胚胎移植妊娠率和产仔率等.结果各试验组胚胎孵化率,Bai组(80.6%)、Lig组(78.3%)极显著高于对照组(51.8%)(P＜0.01).孵化胚胎细胞计数结果显示, Bai组(81.9±6.2)和Lig组(83.9±7.7)与对照组(77.4±5.6)差异极显著(P＜0.01);程序1和2的桑椹胚解冻囊胚发育率,以Bai、Lig组显著高于对照组;其囊胚解冻存活率,2个试验组均好于对照组.受体妊娠率、产仔率以及初生仔鼠、离窝仔鼠平均体重、离窝成活率等指标,各组间均无显著差异(P＞0.05),但以中药有效成分各组别均好于对照组.结果表明中药有效成分Bai和Lig能显著地提高小鼠 2-细胞胚胎体外发育能力,并有利于提高冷冻-解冻后移植胚胎的发育潜力.     

Transfer of Beef Embryos in Dairy Cows: Influence of Recipient and Embryo Quality on Pregnancy Rate and Calving Performance

Contents
On three experimental farms 205 deep frozen Blonde d'Aquitaine embryos were transferred into Holstein dairy cows 7 days after a spontaneous heat. The aim of the study was firstly to examine the influence of recipient and embryo quality on pregnancy rate and secondly to study the calving performance of dairy cows carrying beef embryos. Recipients were divided into three groups based on the quality of the corpus luteum (unsuitable, moderately suitable and suitable) established by rectal palpation. Embryos classified as fair, good and excellent, were randomly distributed over the recipients, including those classified as being unsuitable. The overall pregnancy rate was 36% (standard deviation (SD) = 3.3), dystocia occurred in 48% of births (SD = 5.9), gestation was 297 days (SD = 1.2) and birth weight was 52 kg (SD = 2.0). No statistical differences were found in pregnancy rates and calving performance between the groups of recipients or between the classes of embryos. It is concluded that there is no justification to reject recipients on the basis of the quality of the corpus luteum. Furthermore there is no reason to reject fair embryos for transfer. Beef embryos in dairy cows resulted in much dystocia because of high birth weight, and in long gestation periods.     

Influence of Metabolic Status and Diet on Early Pregnant Equine Histotroph Proteome: Preliminary Findings

Insulin resistance (IR) is characterized by an increase in biomarkers of systemic inflammation and susceptibility to laminitis in horses. Impacts on reproduction include a lengthened interovulatory period in horses. Dietary omega-3 (docosahexaenoic acid [DHA]) promotes anti-inflammatory processes, has been implicated in health benefits, and can reduce cytokine secretion. This preliminary study investigated the impact of IR as well as the influence of dietary supplementation (DHA) on the uterine fluid proteome in early pregnant horses. Mares were artificially inseminated; uterine fluid and embryos were collected on d 12.5 after ovulation. Uterine fluid was pooled for metabolic and diet categories (n = 8; n = 2 per metabolic and dietary status) and concentrated, and the proteome was analyzed using tandem mass spectrometry (iTRAQ). Five proteins met differential abundance criteria (±1.5-fold change, P < .05) in all comparisons (Control C, IS vs. C, IR; C, IS vs. DHA, IS; C, IR vs. DHA, IR). Serum amyloid A, afamin, and serotransferrin were upregulated in C, IR mares but downregulated in DHA, IR mares when compared to C, IS and C, IR, respectively. Quantitative PCR supported mass spectrometry results. The presence of serum amyloid A and serotransferrin in histotroph of IR mares potentially indicates an inflammatory response not seen in IS counterparts. These preliminary findings provide novel evidence on the potential impact of insulin resistance and DHA supplementation on the secreted equine uterine proteome during early pregnancy.     

氨基酸和半胱胺对绵羊胚胎体外发育的影响

将从绵羊卵巢采集的卵母细胞成熟培养22~24h;成熟卵母细胞去除颗粒细胞,选择排出第一极体的卵母细胞,用5μmol/L A23187(5min)联合2mmol/L 6-DMAP(4h)进行孤雌激活;激活后的卵母细胞在培养液中进行培养。研究氨基酸和半胱胺对绵羊胚胎早期体外发育的影响。结果显示:(1)氨基酸能促进胚胎体外发育,0~24h培养添加抑制卵母细胞的卵裂,24h后培养添加比全过程培养添加更能促进胚胎体外发育;(2)24~72h培养添加必需氨基酸(EAA),能促进胚胎体外发育;(3)半胱胺添加量0~100μmol/L,随浓度增高各项发育指标呈增高趋势,添加100~200μmol/L,随浓度增高各项发育指标呈下降趋势。