In vitro production of bovine embryos using sex-sorted sperm.

The objective of this study was to investigate the suitability of sex-sorted sperm for producing viable in vitro embryos for subsequent transfer into recipient cows and heifers on commercial dairy farms. From August 2002 to June 2003, ovaries were collected from 104 producer-nominated Holstein donor cows on seven Wisconsin farms via colpotomy or at slaughter. Oocytes (N=3526) were aspirated from these ovaries, fertilized 22+/-0.

In vitro production of Holstein embryos using sex-sorted sperm and oocytes from selected cull cows.

The objective of this study was to explore potential synergies between sex-sorted sperm and in vitro embryo production for generating replacement heifers on commercial dairy farms. Selected involuntary cull cows (i.e.

Meiotic state of bovine oocytes is regulated by interactions between cAMP, cumulus, and granulosa.

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro.

Possible mechanism for acceleration of meiotic progression of bovine follicular oocytes by growth factors in vitro.

The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles.

A combination of EGF and IGF-I accelerates the progression of meiosis in bovine follicular oocytes in vitro and fetal calf serum neutralizes the acceleration effect.

The effects of a combination of EGF and IGF-I (GFs) on the progress of meiosis and on their developmental competence were examined in cumulus-enclosed bovine oocytes. Exposure to GFs in serum-free, 0.3% PVP-containing maturation medium significantly (P<0.

Factors determining competence of in vitro produced cattle embryos.

Concerns have developed in regard to problems associated with pregnancies and calves produced after use of cattle blastocysts made in the laboratory for embryo transfer. For both empirical studies and commercial purposes, there is a need for assurance that the product of these biotechnologies results in a normally functioning entity of its kind. Ability to use more genetic material from a donor female and in producing blastocysts needs to be improved to increase the efficiency of utilizing in vitro biotechnologies in animal production agriculture and for biomedical purposes.

Efficacy of timed embryo transfer with fresh and frozen in vitro produced embryos to increase pregnancy rates in heat-stressed dairy cattle.

Our objective was to determine whether pregnancy rates in heat-stressed dairy cattle could be enhanced by timed embryo transfer of fresh (nonfrozen) or frozen-thawed in vitro-derived embryos compared to timed insemination. Ovulation in Holstein cows was synchronized by a GnRH injection followed 7 d later by PGF2 alpha and a second treatment with GnRH 48 h later. Control cows (n = 129) were inseminated 16 h (d 0) after the second GnRH injection.

Aspects of follicle and oocyte stage that affect in vitro maturation and development of bovine oocytes.

Success of in vitro maturation (IVM) and production of bovine embryos as related to aspects of follicle source and oocyte size were evaluated. First, it was determined that bovine oocytes continue growing in all follicular sizes studied, including >1- to 15-mm follicles. Populations of oocytes were collected from surface visible (peripheral) and cortical follicles from the same ovaries.

Maintenance of meiotic arrest by increasing [cAMP]i may have physiological relevance in bovine oocytes.

Invasive adenylate cyclase (iAC) reversibly inhibits spontaneous maturation of cumulus-enclosed bovine oocytes by increasing the intracellular concentration of cAMP, [cAMP]i. In this study, physiological aspects of maintaining meiotic arrest in bovine oocytes by iAC were investigated. The maintenance of germinal vesicle arrest by iAC in both cumulus-enclosed and denuded bovine oocytes was concentration dependent (r2 = 0.

Maintenance of bovine oocytes in prophase of meiosis I by high [cAMP]i.

The effects of high intracellular cAMP concentrations ([cAMP]i) on germinal vesicle maintenance of bovine cumulus-oocyte complexes were investigated, using 8-bromo-3',5'-cAMP (8-Br-cAMP) or an invasive adenylate cyclase from Bordetella pertussis to increase the [cAMP]i. The effects of interactions of these agents with macromolecular supplements in culture medium (fetal calf serum, FCS; polyvinylpyrrolidone, PVP; BSA), and different methods of processing complexes before culture, on subsequent germinal vesicle maintenance by invasive adenylate cyclase were studied. While 8-Br-cAMP was unable to maintain germinal vesicle arrest in the majority of oocytes for 20 h (36% with FCS, 24% with BSA, 18% with PVP), it maintained germinal vesicle arrest in a high proportion of cumulus-enclosed oocytes when BSA or PVP was used (37% with FCS, 52% with BSA, 53% with PVP).

Activation of murine oocytes with Ca2+ ionophore and cycloheximide.

A Ca2+ ionophore (A23187, 3 microM) and inhibitor of protein synthesis (cycloheximide, 10 micrograms/ml) were used sequentially as a unique method for activating mouse oocytes in vitro. Brief exposure of oocytes to A23187 followed by 6 hr in cycloheximide resulted in a higher activation rate (93.8%) compared to A23187 or cycloheximide alone (37.

Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion.

We have examined the response of bovine oocytes matured in vitro for 24 hr to parthenogenic activation using compounds that increase intracellular calcium (ionomycin) or inhibit protein phosphorylation (6-dimethylaminopurine, DMAP). Treatment with ionomycin alone caused resumption of meiosis (57.8 +/- 7.

Gene transfer in bovine blastocysts using replication-defective retroviral vectors packaged with Gibbon ape leukemia virus envelopes.

With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat beta-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli beta-galactosidase marker gene in bovine target cells.

Control of germinal vesicle breakdown in bovine x murine hybrid oocytes.

Bovine oocytes cultured in control medium or in medium containing dibutyrylcyclic adenosine monophosphate (dbcAMP) or an inhibitor of cyclic nucleotide phosphodiesterase (3-isobutyl-1-methylxanthine, IBMX) undergo germinal vesicle breakdown (GVBD). On the other hand, mouse oocytes remain arrested at the germinal vesicle (GV) stage when dbcAMP or IBMX is present. When 1 bovine GV stage oocyte is fused to 1 GV stage mouse oocyte, dissolution of both species GV occurred in dbcAMP-supplemented medium.

Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes.

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.

In vitro fertilization and development of bovine oocytes matured in serum-free medium.

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.

Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes.

In cows, protein synthesis is required for germinal vesicle breakdown (GVBD). This study examines more closely the need for protein synthesis and the nuclear changes in the bovine oocyte during 24 h of culture. Bovine oocytes with compact and complete cumulus were washed and incubated in groups of 10 for up to 24 h in 50-microliters drops of TCM-199 supplemented with follicle-stimulating hormone (NIAMADD, 0.

The culture of bovine oocytes to obtain developmentally competent embryos.

Bovine cumulus-oocyte complexes (COC) (n = 4230) were used in this study to assess the effects of culture method, hormonal supplementation, and cumulus cell concentration on maturation, fertilization and development of resulting embryos. Five treatments were evaluated. 1) 10 COC/50-microliter drops under oil in TCM 199 supplemented with 10% heat-treated fetal calf serum, follicle-stimulating hormone (0.

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.

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%).

Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes.

Bovine serum albumin (BSA) and fetal calf serum (FCS) were evaluated as protein supplements for in vitro maturation and fertilization of oocytes from cows and hamsters. BSA and low doses of FCS (0.1 or 1.

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.

Fertilization potential of follicular oocytes classified by stage of cycle and size of follicle.

Bovine follicular oocytes, collected from two sizes of vesicular follicles and from donor animals from three stages of the estrous cycle, were matured and fertilized in vitro. Frequency of fertilization and ability to form male pronuclei after in vitro maturation were found to be independent of both estrual stage and follicular size.