Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer

Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.     

Birth of large calves that developed from in vitro-derived bovine embryos

High birth weights were observed in calves that developed from bovine embryos produced by in vitro maturation (IVM) and in vitro fertilization (IVF) procedures. After IVM and IVF, embryos were either co-cultured in vitro with oviductal epithelial cells or transferred into the sheep oviduct for development to the blastocyst stage. Blastocysts were transferred to the reproductive tracts of recipient heifers and cows for development to term. Birth weights and gestation periods were compared between calves that developed from in vitro-derived embryos and calves born after artificial insemination (AI) of cows in the herd from which recipient females were selected. Gestation periods were not different among the groups (P > 0.05), but calves that developed from IVM/IVF-derived embryos co-cultured in vitro were larger at birth than calves born from IVM/IVF-derived embryos that developed into blastocysts in the sheep oviduct and calves born from AI (P < 0.001). Dystocia and calf mortality were associated with large calf size at birth. These data were collected from an experiment designed for other purposes, and confounding variables and small sample size could have influenced the observed differences in birth weights. Nevertheless, the extreme birth weights of some calves suggest that abnormal prenatal growth occurs in some IVM/IVF-derived bovine embryos and that conditions for co-culture to the blastocyst stage may exacerbate the problem.     

Histology of a heifer placentome after interspecies transfer of a gaur embryo

Histological examination of a single placentome recovered from a Holstein heifer (Bos taurus ) after delivery of a dead 9.5-mo-old gaur (Bos gaurus ) calf revealed failure of proper development of the chorioallantoic villi after interspecies embryo transfer. Macroscopically, the placentome appeared normal in size, its surface was rough, and on crosssection, the penetration of the villi was irregular and very different from the homogeneous penetration in developed cow placentomes. Microscopically, the heifer caruncle had an extensive system of maternal crypts but the villi failed to branch completely and entered only about one half of the available crypt spaces. The epithelial lining of the maternal crypts in the heifer placentome was nearly missing, which was rather unusual for a species of the genus Bos . The overall picture suggested a decreased feto-maternal compatibility, resulting in poor development of the utero-placental contact, retarded fetal growth, and ultimate fetal death.     

DNA fingerprinting of individual species and intergeneric and interspecific hybrids of genera Bos and Bison,subfamily Bovinae

Genome fingerprinting with a hypervariable minisatellite sequence of phage M13 DNA was used to study the genetic variation in individual species of the genera Bos and Bison (subfamily Bovinae) and in their interspecific and intergeneric hybrids. DNA fingerprints were obtained for domestic cow Bos taurus primigenius, vatussy Bos taurus macroceros, banteng Bos javanicus, gaur Bos gaurus, wisent Bison bonasus, bison Bison bison, and for the interspecific and intergeneric hybrids. Compared with the original species, most hybrids showed a greater variation in number and size of hybridization fragments. An association was revealed between the number of hybridization fragments and blood composition of interspecific hybrids resulting from unique crossing of domestic cow and banteng. Pairwise similarity coefficients were calculated to construct a dendrogram of genetic similarity, which reflected the relationships between the parental species and hybrids varying in blood composition. The applicability of the method for identifying interspecific and intergeneric hybrids and for studying the consequences of distant hybridization in the subfamily Bovinae is discussed.     

Phylogenetic relationships of Malayan gaur with other species of the genus Bos based on cytochrome b gene DNA sequences

The Malayan gaur (Bos gaurus hubbacki) is one of the three subspecies of gaurs that can be found in Malaysia. We examined the phylogenetic relationships of this subspecies with other species of the genus Bos (B. javanicus, B. indicus, B. taurus, and B. grunniens). The sequence of a key gene, cytochrome b, was compared among 20 Bos species and the bongo antelope, used as an outgroup. Phylogenetic reconstruction was employed using neighbor joining and maximum parsimony in PAUP and Bayesian inference in MrBayes 3.1. All tree topologies indicated that the Malayan gaur is in its own monophyletic clade, distinct from other species of the genus Bos. We also found significant branching differences in the tree topologies between wild and domestic cattle.     

High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos indicus cows

Two experiments were conducted to assess the effects of environmental temperature and humidity on the quality and developmental capabilities of bovine oocytes. In Experiment 1, Bos taurus (Holstein and crossbred Angus) cows were subjected to 5 weekly sessions of ultrasound-guided follicle aspiration from February 16 through March 23 (cool season) and 5 sessions from May 22 through June 20 (hot season). In Experiment 2, Bos taurus (Holstein) and Bos indicus (Brahman) cows were superstimulated (Super-Ov) during the months of August (hot season) or January (cool season), and each cow was subjected to a single oocyte aspiration session. In each experiment, oocytes were classified as normal or abnormal based on ooplasm morphology and cumulus cell layers. In Experiment 1, oocytes classified as normal were in vitro matured and fertilized (IVM/IVF), and the resulting embryos cultured for 8 d. All oocytes recovered from superstimulated cows in Experiment 2 were matured and fertilized in vitro and the subsequent embryos cultured for 8 d, regardless of their morphological appearance. In Experiment 1, Bos taurus cows produced a higher (P = 0.02) percentage of normal oocytes during the cool season (75.9 +/- 8.0) than during the hot season (41.0 +/- 9.5). The percentage of fertilized oocytes developing to the 2-cell (82.4), 8-cell (65.4) and morula (46.6) stages were also greater (P < or = 0.06) during the cool season than the hot season (45.0, 21.2, 6.0 for 2-cell, 8-cell and morula stages, respectively). In Experiment 2, Bos taurus cows (Holstein) had a lower (P = 0.01) percentage of normal oocytes in the hot season (24.5 vs 80.0) and a lower (P < or = 0.003) percentage of fertilized oocytes developing to the 8-cell, morula and blastocyst stages. No difference (P > or = 0.57) in the percentage of normal oocytes or in embryo development was detected between seasons in Bos indicus (Brahman) cows. In conclusion, high environmental temperature and humidity resulted in a marked decline in the quality of oocytes retrieved from Bos taurus cows and markedly decreased their in vitro developmental capabilities. In contrast, a high percentage of oocytes retrieved from Bos indicus cows exhibited normal morphology and yielded a high proportion of blastocysts, regardless of season.     

Fetal development and gestational changes in Bos taurus and Bos indicus genotypes in the tropics

Weights of the gravid uterus and fetus as well as the fetal measurements were determined at slaughter for 107 Bos taurus cows grazed on improved pastures and for 70 Bos indicus cows grazed on native pastures in northern Australia. The stage of gestation was assessed from palpation per rectum in early-to-mid gestation and at slaughter and from fetal development characteristics at slaughter. The age and breed of the cow and the sex of the fetus did not significantly affect any of the uterine components or fetal measurements. Growth curves had dominant, positive linear components but negative quadratic ones, which improved the fit, particularly for the later stages of gestation. Uterine components and fetal measurements were highly correlated (0.94 to 0.99). For Bos taurus cows, there were higher estimates at birth for weights of the gravid uterus and the fetus, but estimates for other fetal measurements were similar to those for Bos indicus cows. Major fetal growth occurred during the third trimester, with the length of the foreleg tending to change relatively slowly and the head width quite fast during the first trimester. Correction factors for cow liveweight to adjust to commonality for non-pregnancy were 5, 7, 10, 14, 20, 29, 43 and 65 kg for Bos taurus and 2, 4, 6, 10, 15, 23, 35 and 51 kg for Bos indicus at 2 to 9 months of gestation.     

Induction of parturition in Zebu-cross recipients carrying in vitro-produced Bos indicus embryos

Induction of parturition has been used as a management tool in cattle in several countries, but not commonly in Zebu breeds in tropical production systems. When timed according to the stage of gestation, most induction protocols employing a combination of PGF2alpha and a potent, short-acting corticosteroid, resulted in a predictable interval from induction to calving, with no detrimental effects on calf viability; however, the incidence of placental retention was usually elevated. Pretreatment with a long-acting corticosteroid induced placental maturation and greatly reduced the incidence of placental retention following induction with PGF2alpha and a short-acting corticosteroid. Recently, Brazilian cattle breeders have faced a new challenge with a large number of in vitro-produced embryos. Without a reliable method of cryopreservation, large numbers of embryos have been transferred fresh, creating a new demand for protocols for synchronizing recipients and managing their calving. A parturition-induction protocol, efficacious in Bos taurus cattle, was modified for use in Bos indicus cattle (which generally have a longer gestation than B. taurus cattle). Zebu-cross recipients carrying Nelore in vitro-produced embryos were pretreated with 1 mg/60 kg triamcinolone acetonide on Day 280 of gestation, followed by treatment with 500 microg of cloprostenol and 25 mg of dexamethasone on Day 287. The interval from treatment to calving was predictable and the incidence of retained placenta was low, similar to that described previously for B. taurus cattle, demonstrating that this treatment protocol could be used for induction of parturition in Zebu cattle in Brazil.     

Influence of the breed of bull (Bos taurus indicus vs. Bos taurus taurus) and the breed of cow (Bos taurus indicus, Bos taurus taurus and crossbred) on the resistance of bovine embryos to heat

In vitro studies have shown that Bos taurus indicus (B. t. indicus) embryos submitted to heat shock at early stages of development are better able to survive as compared to Bos taurus taurus embryos. Embryo genotype influences resistance to heat shock thus leading to the question as to whether embryos sired by thermo-tolerant breeds exhibit the same resistance to heat shock. In the present study the influence of both oocyte and semen, on the resistance to heat shock (HS) at early stages of in vitro development, was assessed in B. t. indicus [Nelore (N) breed], B. t. taurus [Holstein (H) and Angus (A) breeds] and crossbreds. In Experiment 1, Nelore and crossbred oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from Nelore and Angus bulls. Presumptive embryos were collected and randomly assigned to control (39 degrees C) or HS at 12, 48 or 96 h post insemination (hpi; 41 degrees C for 12h) treatments. The cleavage rates and proportion of embryos developing to the blastocyst and hatched blastocyst stages were recorded on Days 2, 8 and 10, respectively. Heat shock treatment decreased development of both Nelore and crossbred embryos. There was a significant interaction between time (12, 48 or 96 hpi) and temperature for blastocyst rates, i.e., the embryos became more thermotolerant as development proceeded. In Experiment 2, oocytes from Nelore and Holstein cows were fertilized with semen from bulls of either Nelore or Angus breeds, and subjected to 12 h HS at 96 hpi. Heat shock at 96 hpi, decreased embryo development. Additionally, cowxtreatment and bullxtreatment interactions were significant for blastocyst rates, i.e., both breed of cow and breed of bull affected the decline in blastocyst rate caused by heat shock treatment. In conclusion, the present results indicate that Nelore embryos (indicus) are more resistant to heat shock than Holstein (taurus) at early stages of in vitro development, and that embryos become more thermo-tolerant as development proceeds. Additionally, the resistance to heat shock was a result of the genetic contribution from both oocyte and spermatozoa.     

Heterologous fertilization to characterize spermatozoa of the genus Bos

Advances in assisted reproductive techniques, specifically, development of protocols for production of in vitro matured, fertilized and cultured domestic bovine embryos, offer opportunities to apply these techniques to nondomestic bovidae in species preservation. Domestic bovine oocytes were inseminated with nondomestic bovine spermatozoa. Effects of heparin concentration, sperm concentration and their interaction on total and normal in vitro fertilization rates and on subsequent embryo development were evaluated. In different replications, semen from 3 Bos bison, 2 Bos gaurus, 1 Bos grunniens, and 1 Bos javanicus bulls was used. Treatment of spermatozoa included 2 heparin levels (2 and 8 micrograms/mL) and 3 sperm concentrations (1, 3 and 5 x 10(6)/mL). The B. grunniens bull exhibited excessive polyspermy in all treatments; therefore, 1 replicate was completed using 2 levels of heparin (0 and 1 microgram/mL) and 2 sperm concentrations (1 and 2 x 10(6)/mL). After 18 to 22 h, cumulus cells were removed from presumptive zygotes, and a portion thereof was compressed between a slide and coverslip and fixed in acetic acid:ethanol solution. Light microscopy was used to visualize pronuclei and the second polar body as a determinant of fertilization. Remaining presumptive zygotes were placed into embryo culture medium, and blastocyst development was assessed on Days 7 and 8 (fertilization = Day 0). Percentages of total and normal fertilization and of blastocyst formation were analyzed by a logistic regression model, isolating effects due to bull, heparin and sperm concentration, and to their interaction. Work presented here suggests that, just as in Bos taurus, the nondomestic bulls in the Bos species seem to have individual heparin and sperm concentration requirements for successful IVF. We conclude that each bull, domestic or nondomestic, needs to be evaluated individually. Preliminary sperm characterization using domestic cattle oocytes would result in a greater potential for generating purebred embryos of the desired species should scarce female gametes become available.     

从细胞色素b基因全序列探讨大额牛的分子系统发生

大额牛是一种半野生半家养的珍稀牛种, 有关其起源和系统地位一直存在争议。通过PCR扩增、测序等步骤共获得了11头大额牛细胞色素b(Cyt b)基因全序列(1 140 bp)。应用分析软件, 对大额牛11条Cyt b序列进行了分析, 并结合GenBank中牛属动物6个近缘种的同源序列, 以亚洲水牛(Bubalus bubalis)为外群, 分别采用邻接法(NJ)和最大简约法(MP)构建了牛属动物分子系统发育树。序列分析结果表明, 11条大额牛Cyt b序列1 140位点中, 共发现95个变异位点(占分析位点总数的8.33 %), 定义了6种单倍型, 表明大额牛群体的Cyt b基因遗传多态性比较丰富。构建的NJ和MP分子系统树均显示, 大额牛研究群体明显分为3支, 第1支与普通牛(Bos taurus)相聚, 第2支与瘤牛(Bos indicus)相聚, 第3支与印度野牛(Bos gaurus)相聚。系统发育分析表明, 大额牛很可能是印度野牛的家养型或驯化种, 我国大额牛群体可能曾受到其他牛种血缘的入侵。     

Karyotype analysis of mithun (Bos frontalis) and mithun bull x Brahman cow hybrids

We examined the cytogenetics of mithun (Bos frontalis), a domesticated version of the Asian gaur, and hybrids (F(1) generation) produced by artificial insemination of Brahman cows (Bos indicus) with mithun semen. Reproductive potential was also examined in the F(1) generation and a backcrossed heifer for utilization of heterosis. Metaphase chromosome spreads were examined by conventional staining and fluorescence in situ hybridization hybridized with the entire chromosome 1 of mithun as a specific probe. Chromosome 1 of mithun was found to be equivalent to Bos taurus chromosomes 2 and 28. The karyotype of the female mithun (N = 4) comprised 58 chromosomes, including 54 acrocentric and four large submetacentric chromosomes, without the four acrocentric chromosomes found in the domesticated species B. indicus. However, one of the four female mithuns with a normal mithun phenotype had an abnormal karyotype (2n = 59), indicating introgression from B. taurus or B. indicus. The F(1) karyotypes (N = 6, 3♂3♀) of the mithun bull × Brahman cow cross had 2n = 59, intermediate between their parents; they were consistent heterozygous carriers with a centric fusion involving rob(2;28), as expected. Two pronounced red signals were seen in the mithun karyotypes, three red signals in the mithun × Brahman hybrids, and four red signals in the Brahman cattle, in good agreement with centric fusion of bovine rob(2;28). The female backcross hybrid (N = 1) with 2n = 59 had a similar chromosome configuration to the F(1) karyotypes and had rob(2;28). Such female backcross hybrids normally reproduce; however, the F(1) bulls (N = 3) had not yet generated normal sperm at 24 months.     

Developmental competence of oocytes from prepubertal Bos indicus crossbred cattle

The aim of the present study was to evaluate the effect of age on embryogenic competence of oocytes recovered from Bos indicus crossbred calves and heifers. Cumulus-oocyte complexes (COCs) were collected from 4- to 7-month-old calves (experiment 1) and from 9- to 14-month-old heifers (experiment 2) during processing at an abattoir. In both experiments cow COCs were used as control. COCs were in vitro matured and fertilized, and the presumptive zygotes co-cultured with cumulus cells until 224 h post insemination (hpi). In experiment 1, the development rate during the first 68-72 hpi was similar (P > 0.05) between embryos derived from calves and cows. Fewer embryos from calves developed to the blastocyst stage, resulting in a lesser blastocyst production as well as lesser hatching rate (P < 0.05). The embryo development after blastocyst stage was, nevertheless, similar (P > 0.05) between blastocysts derived from calves and cows, suggesting that the development after blastocoele formation is not compromised in embryos derived from calves. In experiment 2, there were no differences (P > 0.05) on cleavage, blastocyst and hatching rates between embryos derived from prepubertal heifers and cows. The rate of blastocyst development until hatching was also similar (P > 0.05). These results indicate that oocytes from 9- to 14-month-old B. indicus crossbred heifers have the same developmental competence as oocytes derived from cows, while ocytes derived from 4- to 7-month-old B. indicus crossbred calves are less competent in developing to the blastocyst stage in vitro. It suggests that oocyte competence in B. indicus crossbred cattle is achieved around 9-14 months of age.     

Production of live calves derived from embryonic stem-like cells aggregated with tetraploid embryos

To date, cloned farm animals have been produced by nuclear transfer from embryonic, fetal, and adult cell types. However, mice completely derived from embryonic stem (ES) cells have been produced by aggregation with tetraploid embryos. The objective of the present study was to generate offspring completely derived from bovine ES-like cells. ES-like cells isolated from the inner cell mass of in vitro-produced embryos were aggregated with tetraploid bovine embryos generated by electrofusion at the 2-cell stage. A total of 77 embryo aggregates produced by coculture of two 8-cell-stage tetraploid embryos and a clump of ES-like cells were cultured in vitro. Twenty-eight of the aggregates developed to the blastocyst stage, and 12 of these were transferred to recipient cows. Six calves representing 2 singletons and 2 sets of twins were produced from the transfer of the chimeric embryos. Microsatellite analysis for the 6 calves demonstrated that one calf was chimeric in the hair roots and the another was chimeric in the liver. However, unfortunately, both of these calves died shortly after birth. Two of the placentae from the remaining pregnancies were also chimeric. These results indicate that the bovine ES-like cells used in these studies were able to contribute to development.     

Embryo transfer as a means of controlling viral infections. III non transmission of bluetongue virus from viremic cattle

Ten holstein heifers were made viremic by inoculation with type 17 or type 18 bluetongue virus (BTV), superovulated, bred artificially with semen from a BTV seronegative bull and slaughtered for the collection of 8-day embryos. A total of 28 embryos were transferred into 28 BTV seronegative recipients.Fourteen transfers resulted in pregnancies. None of the recipients developed BTV antibody during pregnancy or within 30 days of parturition. No antibody or virus was detected in the 14 calves at parturition (of which 4 were lost due to dystocia), in one recumbent calf at the time of euthanasia at 5 days of age or in the remaining 9 healthy calves at 30 days of age. This study suggests that BTV-free calves can be obtained from infected dams by embryo transfer.     

以Cytb基因部分序列分析大额牛系统地位及其种群现状

对12头大额牛Cytb基因部分序列447bp进行分析,共发现33个变异位点,定义了4种单倍型;结合GenBank中牛属普通牛、瘤牛、牦牛和印度褐牛4个近缘种的Cytb基因同源区序列进行分析,以水牛作为外群,分别采用邻接(NJ)法和最大简约(MP)法构建分子系统发育树,得到基本一致的拓扑结构,结果表明大额牛是独立于普通牛、瘤牛和印度褐牛之外的Bos属的一个独立的种。同时还指出目前我国有相当比例的大额牛受到外来物种的入侵,我国的大额牛保护工作正面临非常严峻的考验。     

Superovulation, collection and transfer of embryos and demi-embryos from Boran(Bos indicus ) cows and heifers

Twenty-three Boran(Bos indicus ) cows and heifers were superovulated with pregnant mare serum gonadotropin (PMSG); a total of four embryos and 4.1 +/- 0.3 (mean +/- SEM) ova per ova-producing donor resulted. Follicle stimulating hormone (FSH-P) was then used to superovulate 49 Boran cows for a total of 106 superovulations, of which 63 (59.4%) produced an average of 3.7 +/- 0.4 (mean +/- SEM) embryos. The embryo production was not influenced by either the season or the number of times(one to five) the cows were superovulated. A higher pregnancy rate was obtained when the selection of Boran recipients was based on their plasma-progesterone values (overall 52.5%, single embryos 63.3%, twin demi-embryos 45.8%) than when they were selected by palpation per rectum only (overall 43.8%, single embryos 50%, twin demi-embryos 36.4%). The twinning rate of twin demiembryos was 62.5%, whereas only single calves were born after transfer of two embryos per recipient. No pregnancies were produced following transfer of twin demi-embryos without zonae pellucidae. Transferring single demi-embryos gave a low pregnancy rate (13.3%). Twelve donor Boran cows (21 superovulations) bred with their fathers resulted in a high rate of early embryonic death; additionally, only 20.9% (overall) of the recipients became pregnant. Estrus synchronization of Boran cows with a progesterone releasing intravaginal device (PRID) for a short period (7 d) combined with one injection of prostaglandin (Day 6) produced a larger number of good quality recipients (70.5%) than using double prostaglandin injections (60%).     

Use of F1 progeny of HolsteinxZebu cross cattle as oocyte donors for in vitro embryo production and gene microinjection

This study was designed to determine the possibility of using F1 crossbreed cattle (Holstein x Zebu) as donors of oocytes for in vitro fertilization (IVF) and for pronuclear gene microinjection into in vitro-produced embryos. In the first part of the experiment oocytes from Bos taurus (Holstein), Bos indicus (Zebu) and F1 crossbred Bos taurus x Bos indicus (Holstein x Zebu) genotypes were inseminated with Bos taurus (Holstein) semen and were allocated for in vitro embryo production using conventional IVF procedures. No differences were observed on the in vitro maturation (IVM) rates between breeds (Holstein x Holstein:85%, Zebu x Holstein:84% and Zebu x Holstein x Holstein:88%). Holstein cows yielded the highest number of cumulus oocyte complexes (6.8 per ovary) for in vitro maturation, differing (P<0.05) from Zebu x Holstein and Zebu x Holstein x Holstein F1 by 5.1 and 5.8, respectively. However, the Holstein breed also yielded the lowest percentage of cleavage (45.1 vs 71.9% for Zebu x Holstein and 65.1% for Zebu x Holstein x Holstein). Of the 3 genotypes, the hybrid F1 breed was the most efficient source of oocytes for the production of embryos capable of reaching morulae and blastocyst stages (76 250 ; P< 0.001). In the second part of the study, 599 oocytes from the F1 breed were fertilized in vitro, 1 group of 150 oocytes was used for the determination of the optimal pronuclear visualization period. The highest number of oocytes with 2 pronuclei was observed between 24 to 28 h after IVF (27 to 42%). The remaining 399 oocytes were microinjected with a gene construct bearing the bacterial lacZ gene as the reporter for gene expression. Survival of embryos to microinjection was 73.8%, and 45.5% of them (50 110 ) cleaved in culture. Of the microinjected embryos, 1 out of 50 showed beta-galactosidase activity. These findings indicate that a tropical crossbreed of cattle (Zebu x Holstein x Holstein) can be used as a source of oocytes for IVF programs and gene microinjection studies.