Death losses due to stillbirth, neonatal death and diseases in cloned cattle derived from somatic cell nuclear transfer and their progeny: a result of nationwide survey in Japan

To obtain the data concerning death losses due to stillbirth, neonatal death and diseases in cloned cattle derived from somatic cell nuclear transfer (SCNT) and their progeny produced by Japanese institutions, a nationwide survey was carried out in July-August, 2006. As a result, lifetime data concerning 482 SCNT cattle (97.5% of cattle produced in the country at that time) and 202 progeny of SCNT cattle were accumulated and the death loss of these cattle was analyzed. Although 1/3 of delivered SCNT calves died during the perinatal period due to stillbirth and neonatal death, incidence of death loss due to diseases in SCNT cattle surviving more than 200 days after birth seems to be the same as these in conventionally bred cattle. In contrast, progeny of SCNT cattle showed the same level in death loss as observed in conventionally bred cattle throughout their lifetime. These results suggest that robust health would be expected in SCNT cattle surviving to adulthood and their progeny.     

A 12-month feeding study of reproduction/development in rats fed meat/milk powder supplemented diets derived from the progeny of cloned cattle produced by somatic cell nuclear transfer

The present 12-month feeding study was carried out with rat groups fed a diet supplemented with meat or milk (meat/milk) derived from the progeny of clones produced by somatic cell nuclear transfer (SCNT) technology. It was conducted to obtain data concerning the chronic toxicities of these edible products during the process of development and reproduction in rats fed such products. The rats were subjected to clinical observations for general health condition and examinations such as sensory/reflex function, grip strength, motor activity, body weight, food consumption, ophthalmology and urinalysis. Moreover, sexually matured rats fed the test diets were mated and examined for items such as the reproductive performances of the dams and health of their pups. After the feeding period, factors related to rat health status, based on the findings for hematology, blood biochemistry, necropsy, organ weight and histology, were examined. There were no biologically significant differences in these factors between the rat groups fed meat/milk powder supplemented diets derived from the progeny and those fed meat/milk powder supplemented diets derived from conventionally bred cattle. Therefore, the present chronic toxicity study suggests that meat and milk derived from the progeny of SCNT cattle might be equivalent to those derived from conventionally bred cattle in use as dietary supplements for rats.     

哺乳动物体细胞核移植胚胎发育中表观重编程的研究进展

体细胞核移植（somatic cell nuclear transfer,SCNT）是一种能将已分化的体细胞重编程为全能胚胎的繁殖生物技术,在良种扩繁、濒危物种保护和治疗性克隆等方面有着广泛的应用前景,但极低的克隆效率、克隆动物胎盘异常、出生后胎儿畸形等严重限制了该技术的实际应用。造成克隆效率低和胚胎发育异常的主要原因是供体核表观遗传重编程错误或不完全。1958年,将非洲爪蟾（Xenopus laevis）幼体肠细胞核移入去核卵母细胞,获得了第1例SCNT动物个体;1986年,通过电融合1个卵裂球与去核卵母细胞成功获得了3只存活的羔羊;1997年,将成年母羊的乳腺上皮细胞与去核卵细胞电融合,获得首个SCNT哺乳动物"多利",开启了克隆时代,目前牛、小鼠、山羊、猪、欧洲盘羊、家兔、家猫、马、大鼠、骡子、狗、雪貂、狼、水牛、红鹿、单峰骆驼、食蟹猴等相继成功克隆,其中最引人瞩目的是2018年食蟹猴的成功克隆。作者通过将SCNT胚胎与受精胚胎的发育进行对比,阐述了SCNT过程中DNA甲基化、组蛋白修饰、基因组印迹、染色体状态等的重编程过程和缺陷,并从表观修饰剂、组蛋白去甲基化酶、抑制Xist表达、补充鱼精蛋白和精子RNA方面探讨单独或联合消除表观遗传重编程障碍对克隆效率的影响。随着低样本量测序技术的发展和完善,人们能够在SCNT胚胎中检测到更详细的全基因组表观遗传修饰图谱,进一步揭示SCNT胚胎表观遗传重编程中的缺陷,为提高克隆效率提供了线索。通过上述内容的阐述,希望为后续开发联合消除多种表观遗传障碍而提高克隆效率的策略和思路。     

Development of genome engineering technologies in cattle: from random to specific

The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure.However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle.     

Health status and productive performance of somatic cell cloned cattle and their offspring produced in Japan

Since the first somatic cell cloned calves were born in Japan in 1998, more than 500 cloned cattle have been produced by somatic cell nuclear transfer and many studies concerning cloned cattle and their offspring have been conducted in this country. However, most of the results have been published in Japanese; thus, the data produced in this country is not well utilized by researchers throughout the world. This article reviews the 65 reports produced by Japanese researchers (62 written in Japanese and 3 written in English), which employed 171 clones and 32 offspring, and categorizes them according to the following 7 categories: (1) genetic similarities and muzzle prints, (2) hematology and clinical chemistry findings, (3) pathology, (4) growth performance, (5) reproductive performance, (6) meat production performance and (7) milk production performance. No remarkable differences in health status or reproductive performance were found among conventionally bred cattle, somatic cell cloned cattle surviving to adulthood and offspring of somatic cell cloned cattle. Similarities in growth performance and meat quality were observed between nuclear donor cattle and their clones. The growth curves of the offspring resembled those of their full siblings.     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.     

Purebred-crossbred performance and genetic evaluation of postweaning growth and carcass traits in Bos indicus x Bos taurus crosses in Australia

Growth and carcass data on 7,154 cattle from a purebred project and 1,241 cattle from a crossbred project, comprising 916 first-crosses and 325 purebred Brahman controls, were analyzed to estimate genetic parameters, including the genetic correlations between purebred and crossbred performance (rpc). The data also allowed the estimation of sire breed means for various growth and carcass traits. Crossbred calves were produced using 9 Angus, 8 Hereford, 7 Shorthorn, 14 Belmont Red, and 8 Santa Gertrudis sires bred to Brahman dams. These same sires produced 1,568 progeny in a separate purebreeding project. Cattle in both projects were managed under two finishing regimens (pasture and feedlot) to representative market live weights of 400 (domestic), 520 (Korean), and 600 kg (Japanese). The traits studied included live weight at around 400 d of age (400W), hot carcass weight (CWT), retail beef yield percentage (RBY), intramuscular fat percentage (IMF), rump fat depth (P8), and preslaughter ultrasound scanned eye muscle area (SEMA). Estimated breeding values (EBV) of sires from their BREEDPLAN genetic evaluations were used to assess their value in predicting crossbred performance. Regressions of actual crossbred calf performance on sire EBV for each of the traits differed little from their expectation of 0.5. Angus sires produced crossbred carcasses with the highest P8 and lowest RBY but highest IMF. In contrast, crossbred progeny from Belmont Red sires had the lightest 400W and CWT, lowest P8, and highest RBY. Estimates of rpc were 0.48, 0.48, 0.83, 0.95, 1.00, and 0.78 for 400W, CWT, RBY, IMF, P8, and SEMA, respectively. Commercial breeders selecting sires for crossbreeding programs with Brahman females, based on EBV computed from purebred data, might encounter some reranking of sire's performance for weight-related traits, with little expected change in carcass traits.     

ASCL2基因在体细胞克隆牛和自然繁殖牛肺脏中的甲基化分析

为探讨ASCL2基因在体细胞克隆牛中的重编程状态,本研究通过亚硫酸氢盐测序法（BSP）对体细胞克隆牛和自然繁殖牛肺脏中ASCL2基因启动子区CpG岛上的CpG位点进行了甲基化状态分析。结果显示,体细胞克隆牛的甲基化水平（28.41%）显著高于正常对照组（7.62%）（P＜0.05）。推测ASCL2基因的异常甲基化可能是造成体细胞克隆牛新生死亡及肺脏发育异常的原因之一。     

Heterosis, maternal and direct effects in double-muscled and normal cattle: I. Reproduction and growth traits

Data from a two-breed group diallel experiment involving double-muscled (DM) and normal (N) cattle were analyzed to evaluate the importance of heterosis, maternal and direct effects for reproduction and growth traits. The DM cattle were from a composite of primarily Angus, Charolais, Galloway and Hereford breeds, and N cattle were crossbred cattle with at least 50% Hereford breeding. The data comprised a total of 491 matings and 389 calvings in four breeding seasons. Records on calving performance, calving date, calf crop born and weaned, sex ratio of progeny and weight of calves were analyzed using least squares procedures. Significant heterosis of 5 to 12% was observed for all the calf crop and growth traits, except for birth weight. Heterosis resulted in 24% reduction in the incidence of dystocia and perinatal calf mortality (P less than .05). The significant heterosis was due mainly to poor production in DM X DM crosses. The DM cows were superior (P less than .05) to N cows in reciprocal crossing (maternal effect) for all the calf crop traits, but had higher incidence of calving difficulty and a higher proportion of male progeny. Significant direct effect was observed for all the traits except calving date and sex ratio among progeny. The N sires were superior for all the calf crop traits and caused less incidence of dystocia and perinatal mortality compared with the DM sires. Significant differences were obtained between the straightbreds for all the traits except calving date. The N straightbreds were superior to the DM straightbreds for all the significant traits.     

Integrating Ranch Forage Production,Cattle Performance,and Economics in Ranch Management Systems for Southern Florida

The presence of grazing cattle near open waterways has created environmental concerns related to the potential for water contamination. In Florida the removal of cattle from grazing landscapes or decreasing stocking density is being investigated as one option to improve the quality of surface water runoff draining into Lake Okeechobee, Florida. The objective of this study was to determine the effects of stocking rate on cow-calf performance, forage availability and quality, and ranch economic performance. Experimental pastures were established on a southern Florida cow-calf operation with stocking rates of 0.58, 1.01, and 1.35 ha·cow^-1 on summer pastures and 0.93, 1.62, and 2.16 ha·cow^-1 on winter pastures, corresponding to high, medium, and low rates, respectively. The study was conducted over 4 consecutive production years. Cow body condition scores (BCS), pregnancy rate, and calf average daily gain were used as measures of animal performance. Forage utilization was estimated by measuring the difference between forage yield inside and outside grazing exclusion cages and forage quality by crude protein and in vitro organic matter digestibility. Forage yield, utilization, and quality were not significantly affected by stocking rate. Although statistically not significant (P = 0.17), cattle in the high stocking rate experienced a numerically greater loss of BCS following the winter grazing period, but stocking rate did not affect pregnancy rate or calf gains. Production (kg weaned calves·ha^-1) was increased (P < 0.01) for a high stocking rate compared with medium and low stocking rates. Overall ranch profitability will decrease as stocking rates decline. Ranch revenues decrease one-for-one as stocking rates decrease. At the same time, unit cow costs increase at an increasing rate as fewer brood cows are left to support the ranch's fixed cost structure.     

Post-Death Cloning of Endangered Jeju Black Cattle (Korean Native Cattle): Fertility and Serum Chemistry in a Cloned Bull and Cow and Their Offspring

To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell nuclear transfer (SCNT) in a previous study. In the present study, we examined the in vitro fertilization and reproductive potentials of these post-death cloned animals. Sperm motility, in vitro fertilization and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee) and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial insemination (AI). There were no differences in sperm motility or developmental potential of in vitro fertilized embryos between the post-death cloned bull and its extinct nuclear donor bull; however, the embryo development ratio was slightly higher in the cloned sperm group than in the nuclear donor sperm group. After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation proceeded normally until day 287. From this post-death cloned sire and dam, a JBC male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic paternity/maternity of the cloned JBC bull and cow with regard to their offspring was confirmed using International Society for Animal Genetics standard microsatellite markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In addition, there were no significant differences in blood chemistry among the post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is the first report showing that a pair of cattle, namely, a post-death cloned JBC bull and cow, had normal fertility. Therefore, SCNT can be used effectively to increase the population of endangered JBC.     

Mucosal disease of cattle: a late sequel to fetal infection

The introduction of a heifer which was persistently infected with bovine virus diarrhoea-mucosal disease virus into groups of pregnant cattle resulted in abortion, neonatal death, persistent infection and, subsequently, mucosal disease in the surviving progeny. Cattle affected with mucosal disease were invariably seronegative at the time of investigation and subsequent cases occurred only in calves previously identified as seronegative and persistently infected. The detection of virus antigen by immunofluorescent staining of cells obtained from the nasopharynx was shown to be an efficient and rapid method for identifying persistently infected cattle, correlating perfectly with virus isolation.     

Genetic parameters for direct and maternal calving ease in Walloon dairy cattle based on linear and threshold models

Calving ease scores from Holstein dairy cattle in the Walloon Region of Belgium were analysed using univariate linear and threshold animal models. Variance components and derived genetic parameters were estimated from a data set including 33 155 calving records. Included in the models were season, herd and sex of calf × age of dam classes × group of calvings interaction as fixed effects, herd × year of calving, maternal permanent environment and animal direct and maternal additive genetic as random effects. Models were fitted with the genetic correlation between direct and maternal additive genetic effects either estimated or constrained to zero. Direct heritability for calving ease was approximately 8% with linear models and approximately 12% with threshold models. Maternal heritabilities were approximately 2 and 4%, respectively. Genetic correlation between direct and maternal additive effects was found to be not significantly different from zero. Models were compared in terms of goodness of fit and predictive ability. Criteria of comparison such as mean squared error, correlation between observed and predicted calving ease scores as well as between estimated breeding values were estimated from 85 118 calving records. The results provided few differences between linear and threshold models even though correlations between estimated breeding values from subsets of data for sires with progeny from linear model were 17 and 23% greater for direct and maternal genetic effects, respectively, than from threshold model. For the purpose of genetic evaluation for calving ease in Walloon Holstein dairy cattle, the linear animal model without covariance between direct and maternal additive effects was found to be the best choice.     

Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development

Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm‐transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro‐fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.     

延边黄牛体细胞核移植的研究进展

牛体细胞核移植(somatic cell nuclear transfer,SCNT)是一套极其复杂的技术体系,它包括卵母细胞的成熟、供核细胞的准备、卵母细胞的显微操作、细胞融合、卵母细胞激活和胚胎培养。因此,许多因素影响着核移植胚胎发育。虽然陆续有克隆牛出生的报道,但是克隆效率依然低下。本综述对延边黄牛体细胞核移植体系的6个方面进行简单综述,以期为探究延边黄牛体细胞克隆的最优化条件、建立最优化的培养体系、提高克隆效率提供参考。     

Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non‐cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non‐cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non‐cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.     

Simple example illustrating adjustment for merit of mates in a national cattle evaluation program

One of the requirements for an effective progeny test is that each sire being evaluated should be mated to a random sample of females; however, random mating is seldom practiced by purebred beef cattle producers. A simple example involving 18 individuals (14 parents and 4 nonparents) was developed to help students enrolled in a junior-level animal breeding course comprehend how current genetic evaluation methodology, used on an industrywide basis, accounts for nonrandom mating by purebred beef cattle breeders for a maternally influenced trait such as calf weaning weight. With this example, students are able to visualize, algebraically, how a sire's direct and maternal genetic values for weaning weight are adjusted for merit of mates, permitting sires to be compared regardless of mating criteria used by purebred beef cattle producers.     

Comparison of the growth performances of offspring produced by a pair of cloned cattle and their nuclear donor animals

In the present study, the growth performance of a calf produced by mating a somatic cell cloned dam and sire was compared with that of its full siblings produced by mating the cattle used as nuclear donors for the cloned animals. The somatic cell cloned dam and sire were derived from cultured cumulus cells and ear cells, respectively. The cloned dam was artificially inseminated with semen from the cloned sire. A female calf was produced that was reared under general group feeding conditions. The calf was subjected to a clinical examination and to hematology, serum biochemistry, and telomere length analyses; all of these tests indicated that the calf was normal. The growth characteristics (body weight and shoulder height) of the calf fell within the range of the full siblings of the same sex produced by mating the animals used as the nuclear donors of clones. These findings suggest that the same breeding performance is expected from mating a cloned dam and sire as from mating the animals used as nuclear donors for the clones.     

Early Pregnancy Diagnosis by Serum Progesterone and Ultrasound in Sheep Carrying Somatic Cell Nuclear Transfer-Derived Pregnancies

Early pregnancy diagnosis and monitoring play an important role following embryo transfer in sheep. The aims of the current study were to investigate (i) the pattern of serum progesterone profiles in sheep carrying somatic cell nuclear transfer (SCNT)‐derived (clone) pregnancies, and (ii) the frequency of pregnancy loss during development following SCNT embryo transfer. Sheep SCNT embryos were made using standard nuclear transfer techniques. Day 7 embryos were surgically transferred to oestrus‐synchronized recipients (n = 27). As a control, normal fertile ewes (n = 12) were bred by natural breeding. Serum was collected from all the ewes on the day of estrus (day 0 sample), 7 days post‐estrus (day 7 sample) and 19 days post‐estrus (day 19 sample) and every 10 days thereafter until lambing or pregnancy loss occurred. Serum progesterone (P4) was assessed using enzyme immunoassay. Pregnancy was confirmed by ultrasound scanning on day 35 of pregnancy followed by subsequent scanning every 10 days. In control ewes, pregnancy rate on day 35 was 83.3% (10/12), whereas in the ewes that received SCNT embryos, it was 22.2% (6/27; p < 0.05). The day 45 pregnancy rate in the control ewes was 83.3%, whereas in the SCNT embryo recipients it was 11.0% (p < 0.05). Hormone analysis revealed that SCNT embryo recipients exhibited a significantly lower P4 profiles at different time points in pregnancy compared to controls (p < 0.05). This study highlights the use of serum progesterone in combination with ultrasound for the investigation of embryo loss and crucial times during development of normal and SCNT embryos in sheep. Further, the serum P4 levels directly reflect the degree of placental development in these two groups.     

Effects of breed proportion and components of heterosis for semen traits in a composite cattle breed

The aim of this study was to estimate the non‐additive genetic effects of the dominance component of heterosis as well as epistatic loss on semen traits in admixed Swiss Fleckvieh, a composite of Simmental (SI) and Red Holstein Friesian (RHF) cattle. Heterosis is the additional gain in productivity or fitness of cross‐bred progeny over the mid‐purebred parental populations. Intralocus gene interaction usually has a positive effect, while epistatic loss generally reduces productivity or fitness due to lack of evolutionarily established interactions of genes from different breeds. Genotypic data on 38,205 SNP of 818 admixed, as well as 148 RHF and 213 SI bulls as the parental breeds were used to predict breed origin of alleles. The genomewide locus‐specific breed ancestries of individuals were used to calculate effects of breed difference as well as the dominance component of heterosis, while proxies for two definitions of epistatic loss were derived from 100,000 random pairs of loci. The average Holstein Friesian ancestry in admixed bulls was estimated 0.82. Results of fitting different linear mixed models showed including the dominance component of heterosis considerably improved the model adequacy for three of the four traits. Inclusion of epistatic loss increased the accuracy of the models only for our new definition of the epistatic effect for two traits, while the other definition was so highly correlated with the dominance component that statistical separation was impossible.