Production and breeding of transgenic cattle using in vitro embryo production technology.

Transgenic technology permits major modifications of phenotype by introducing subtle changes in genotype. For domestic farm species, genetic modification may be used to enhance agricultural production or to generate novel genotypes capable of producing heterologous proteins for biomedical applications. The advent of in vitro embryo production techniques has facilitated the large-scale, commercial use of transgenic technology in cattle. Accordingly, we employed in vitro-produced zygotes and embryos in an effort to generate transgenic cattle. Overall, pronuclei in 36,530 in vitro matured and fertilized zygotes were microinjected with a construct designed to express human alpha-lactalbumin in the mammary gland. Of these, 1,472 developed and were transferred to recipients, including 148 twin transfers. Initial pregnancy rate on Day 30 of gestation was 28% (374/1,324). Subsequent calving rate was 17% (226/1,324). Eighteen calves (8%) were transgenic. In vitro produced embryos were used to facilitate breeding of transgenic bulls. Frequency of transgene transmission varied from 3 to 54% between bulls, indicating varying degrees mosaicism. Embryos produced in vitro by these bulls were biopsied and screened for transgenesis prior to transfer to recipients; so far all (6/6) calves born from screened, transgenic embryos were themselves transgenic.

Nuclear transfer from somatic cells: applications in farm animal species.

The reconstruction of mammalian embryos by transfer of a blastomere nucleus to an enucleated oocyte or zygote allows for the production of genetically identical individuals. This has advantages for research (that is, as biological controls) and commercial applications (that is, multiplication of genetically valuable livestock). However, the number of offspring that can be produced from a single embryo is limited both by the number of blastomeres (embryos at the 32-64-cell stage are the most widely used in farm animal species) and the limited efficiency of the nuclear transfer procedure. The ability to produce live offspring by nuclear transfer from cells that can be propagated and maintained in culture offers many advantages, including the production of many identical offspring over an extended period (since cultured cells can be frozen and stored indefinitely) and the ability to modify genetically or to select populations of cells of specific genotypes or phenotypes before embryo reconstruction. This objective has been achieved with the production of lambs using nuclei from cultured cells established from embryonic, fetal and adult material. In addition, lambs transgenic for human factor IX have been produced from fetal fibroblasts transfected and selected in culture.

Kinetics of first polar body extrusion and the effect of time of stripping of the cumulus and time of insemination on developmental competence of bovine oocytes.

Kinetics of extrusion of the first polar body was examined as well as the effect of the time of stripping of the cumulus cells on this kinetics. In addition, the effects of time of stripping and time of insemination on developmental competence of the oocytes, as evaluated by the percentage of morulae and blastocysts, were studied. Polar body extrusion occurred in 80% of the oocytes between 12 and 18 h after the onset of maturation. The remainder of the oocytes did not extrude a polar body at all. Stripping of the cumulus at 12 h after the onset of maturation delayed polar body extrusion significantly by about 1 h. No significant differences were found in the percentage of oocytes that could be fertilized, and the percentage of oocytes that cleaved and developed to the morula and blastocyst stages, between oocytes that were stripped free of cumulus and inseminated at either 16 or 20 h after onset maturation. Oocytes that had extruded a polar body at either 16 or 20 h after onset maturation showed significantly higher percentages of cleavage and development than oocytes that had not extruded a polar body at those time points. However, the percentage of oocytes that could be fertilized was not affected.

Challenges and progress in the production of transgenic cattle.

The production of transgenic cattle presents a number of unique challenges not encountered in other species. First, the survival of microinjected zygotes is low; only 15% in vivo-derived develop into morulae and blastocysts and, of these, only about 18% yield live calves. Second, transgene integration frequency is relatively low, around 3%. Thus, more than 1000 zygotes must be injected to produce a single transgenic calf. Obtaining sufficient zygotes from donor cattle to sustain a transgenic cattle programme is logistically and financially prohibitive, since the average superovulated donor yields only about four microinjectable zygotes per collection attempt. In vitro oocyte maturation and fertilization techniques may be used to alleviate this problem, although initially the developmental potential of in vitro-derived microinjected zygotes is lower than their in vivo-produced counterparts (8% v. 15%, respectively, yield morulae and blastocysts). Since only 3-5% of calves born from microinjected zygotes produced in either fashion yield transgenics, at least 20-30 pregnancies must be carried to term for every transgenic calf born. These conditions require that large herds of donor and recipient cattle be maintained. Recipient requirements could be reduced if transgene integration frequency could be increased, but improvements in the near future are unlikely since the mechanism of integration after pronuclear microinjection is poorly understood. Alternatively, embryos could be screened for integrated transgenes before transfer; however, efforts in this area have been complicated by high frequencies of false positive results. Although yet to be developed, bovine embryonic stem cells would alleviate many of these problems and permit a wider range of genetic manipulations.

Some factors affecting the efficacy of oviduct tissue-conditioned medium for the culture of early bovine embryos.

Oviduct tissue-conditioned medium was evaluated for the culture of IVM-IVF bovine zygotes to the compact morula (cM) and blastocyst (BL) stages. Development was unaffected (P greater than 0.50) by freezing and thawing of conditioned medium: no. cM + BL/no. cleaved ova obtained after culture in nonfrozen, frozen-thawed, and control treatments were 31/148 (21%), 26/124 (21%) and 5/86 (6%), respectively. The greatest proportion of normal development was obtained after a conditioning period of 48 h (P less than 0.05): no. of cM + BL/no. cleaved ova in media conditioned for 5, 24, 48 and 96 h were 23/114 (20%), 38/112 (34%), 43/115 (37%) and 36/125 (29%), respectively. Development declined with increasing dilutions of conditioned media (P less than 0.005): no. cM + BL/no. cleaved ova for 100, 75, 50, 25 and 0% conditioned medium were 32/94 (34%), 29/94 (31%), 17/82 (21%), 10/94 (11%) and 11/73 (15%), respectively. The oestrous cycle stage from which oviducal tissue was obtained did not affect development (P greater than 0.75); no. cM + BL/no. cleaved ova was 21/63 (33%) at oestrus and 21/79 (27%) in the luteal phase.

Characterization of developmental arrest in early bovine embryos cultured in vitro.

The susceptibility of early bovine embryos to developmental arrest ("blocking") in vitro was examined. Embryos, obtained from superovulated donors, were cultured in vitro in Ham's F10 culture medium or in vivo in sheep oviducts. Treatments were terminated on Day 7 post-donor estrus (estrus = day 0), and the embryos were evaluated for development. Experiment 1 tested whether the 8- to 16-cell block was reversible. One- to two-cell embryos were cultured in vitro to the 8-cell stage (2 d), then in vivo for 3 d; controls were cultured in vitro or in vivo for 5 d. Forty-two percent (19/45) of in vivo controls developed normally; none (0/55; 0%) of the in vitro controls cleaved past the 9- to 16-cell stage. Only 4% (2/48) of the embryos cultured to eight cells in vitro developed normally after culture in sheep oviducts, indicating that the block was irreversible. Irreversibility was not caused by overt cell death, since 33/33 (100%) of blocked embryos responded positively to fluorescein diacetate vital staining. Experiment 2 tested the effect of in vitro exposure at specific cell stages on subsequent in vivo development. Embryos at the 1- to 2-, 3- to 4-, 5- to 8- and 9- to 16-cell stages were assigned randomly to one of the following treatments: in vivo culture; in vitro culture; or 24 h in vitro culture, followed by in vivo culture. Subsequent in vivo development was affected by 24 h of in vitro culture (P<0.05) only in 3- to 4-cell embryos (11/41, 27% vs 22/41, 54% for in vivo controls). We conclude that 1) the block is a manifestation of in vitro exposure during the four- to eight-cell stage, and 2) the block, while irreversible, is not the result of overt embryonic death.

Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium.

In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte.

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Culture of one- and two-cell bovine embryos to the blastocyst stage in the ovine oviduct.

The ovine oviduct was evaluated as a culture system for early bovine embryos. One- to two-cell embryos were collected from superovulated heifers killed 36 or 48 h after the onset of estrus, embedded in agar cylinders, and transferred to oviducts ligated at the uterotubal junction. After 5 d (6.5 to 7.0 d after donor estrus), embryos were recovered and evaluated for development to the late morula or blastocyst stage. In Experiment 1, 86 embryos were cultured in 10 ewes in which the onset of estrus was synchronized with that of the donors. Fifty-eight embryos (68%) were recovered; of these, 31 (53%) had continued normal development. In Experiment 2, development in ovariectomized versus intact cyclic ewes was compared. Recovery from ovariectomized ewes (26/39, 67%) did not differ from intact cyclic ewes (26/35, 74%) and the proportion developing normally also did not differ (ovariectomized: 7/26, 27%; intact cyclic: 11/26, 42%). In Experiment 3, embryo development was compared in anestrous versus ovariectomized ewes. Recovery rate (anestrous: 22/43, 51%; ovariectomized: 20/51, 39%) and the proportion developing normally (anestrous: 8/22, 37%; ovariectomized: 9/20, 45%) did not differ between treatments. Developmental competence of oviduct-cultured embryos was tested by transfer to 16 synchronous heifers, of which eight (50%) became pregnant; five delivered calves. Results indicate that the ovine oviduct provides an adequate site for the culture of early bovine embryos.

Development potential of bovine oocytes matured in vitro or in vivo.

Bovine oocytes matured in vivo or in vitro were evaluated after sperm-oocyte incubation for frequency of sperm penetration, frequency of male pronuclei formation, and embryonic development. The frequency of sperm penetration was not different for in vitro matured oocytes (216/295, 73%) vs. in vivo matured oocytes (119/176, 70%). However, formation of male pronuclei was reduced (p less than 0.05) for oocytes matured in vitro (149/216, 69%) vs. in vivo (104/119, 88%). Early embryonic development was evaluated 48 h after the onset of sperm-egg incubations. In vitro matured and fertilized oocytes failed to develop to the 2-cell stage (3/88, 3%), whereas oocytes matured in vivo showed normal development (23/56, 40%) to the 2- and 4-cell stage. Development to the blastocyst stage was evaluated after 5 days in ovine oviducts (in vivo). Morulae and blastocysts were obtained only after in vitro fertilization from oocytes that were in vivo-matured (recovered from oviduct, 14/56, 25%; recovered from follicle, 36/80, 45%). Oocytes that were matured in vitro and fertilized in vitro failed to develop to morulae (0/33) in vivo.

Bovine in vitro fertilization with frozen-thawed semen.

A procedure to obtain high and repeatable fertilization frequencies for bovine in vitro fertilization (IVF) with frozen-thawed sperm was developed. IVF frequency of in vitro matured oocytes was increased by a swimup sperm separation procedure (P=0.01) or treatment of sperm with the glycosaminoglycan heparin (P=0.0001), but the two factors did not interact (P=0.23). Heparin was the most important factor in increasing IVF frequencies. The fertilization frequency was not affected by the batch of oocytes used (P=0.38), but bull effects were present (P<0.05). Within a bull, the IVF system was highly repeatable and varied between trials no more than +/- 12% in fertilization frequency with an overall fertilization frequency of 299 379 (79%) on four trials over four bulls. In vivo matured oocytes fertilized in vitro were transferred to ewe or heifer oviducts. Morulae or blastocysts were recovered from ewes after four to five days, while conceptuses were present in the bovine after 25 days (diagnosed by ultrasound). Embryonic development from the IVF system either pre- or postimplantation was normal.

A review of ovarian follicular cysts in cows, with comparisons to the condition in women, rats and rabbits.

Some morphological and endocrinological aspects of cystic follicles in cows, women, rats, and rabbits were reviewed. The etiology of follicular cysts remains a mystery, in part due to the difficulty of studying them during formation, particularly in women and cows. Investigations of the morphological and endocrinological nature of cystic follicles during their formation should enhance our understanding of the factors that cause them. Rats and rabbits may provide useful and convenient experimental models for the study of factors which contribute toward cyst formation.

Uterine specific antigens in the ewe.

Uterine flushings from ewes on days 0, 3, 6, 9, 12 and 15 of the estrous cycle were analyzed for total protein content. Flushings from days 9, 12 and 15 had greater (P<.01) amounts of protein than those from 0, 3 and 6. Antisera to uterine fluids from ewes at day 10 to 12 or day 14 to 15 of pregnancy detected two uterine-specific antigens in uterine flushings at day 7, 11 and 15 but not at days 0 and 3 of the cycle. A third uterine antigen was also detected in kidney tissue extracts. All three antigens were present in endometrial extracts at each stage examined. Progesterone, or estrogen plus progesterone, administration to ovariectomized ewes induced the appearance of the two uterine-specific antigens. The third antigen was detectable even in ovariectomized ewes. No pregnancy-specific antigens were detected in flushings from days 7, 11 or 15 of gestation. The effect of pregnancy on endometrial protein synthesis was examined in vitro . No differences were seen in the incorporation of (3)H-leucine in day 11 pregnant or nonpregnant or in day 14 pregnant or nonpregnant endometrium. No differences in total uterine lumenal protein were observed. Endometrial secretions, obtained by conditioning media with endometrial explant cultures, were evaluated to assess their effect on protein synthesis in day 11 embryos cultured in vitro . No significant effects of endometrial secretions or serum were observed.