N.L. First

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.

In vitro development of bovine one-cell embryos: Influence of glucose, lactate, pyruvate, amino acids and vitamins

To elucidate the effect of nutrient substrates on embryo development, in vitro fertilized bovine one-cell embryos were cultured in a medium similar to synthetic oviduct fluid (SOF) but without glucose and containing 3.3 mM lactate, 0.3 mM pyruvate and 3 mg/ml bovine serum albumin (BSA) at 39 degrees C in 5% CO(2) in air. Results indicated that addition of glucose was not only unnecessary, but it also had a deleterious effect on embryo development to the morula stage. Lactate supported embryo development up to the morula stage as well as pyruvate. Supplementation with 20 amino acids contained in basal medium Eagles (BME) and minimum essential medium (MEM) improved development to the morula stage dramatically and increased the cell number compared with that of the controls. Addition of the vitamins from MEM to SOF had no beneficial effect. The SOF with amino acids did not increase the frequency of blastocysts 7 days after in-vitro fertilization but did increase the total number of cells compared with that of the controls. Frequency of blastocysts at Day 7 in SOF with amino acids was equivalent to that of co-culture although the total cell number was lower. These results demonstrate that a semi-chemically defined medium can successfully support the development of bovine embryos to the morula stage to a limited extent, but the medium lacks some nutrients or growth factors to fully support development through the blastocyst stage.

Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro

Glycosaminoglycans (GAGs) were reported to induce acrosome reactions (AR) in epididymal and ejaculated bovine sperm (4,5). The GAGs chondroitin sulfate A (CS-A) and heparin were tested on ejaculated bovine sperm for their ability to increase in vitro fertilization (IVF) frequencies. Regardless of treatment, a sperm-egg incubation time of 18 hr was sufficient to achieve maximal rates of fertilization. The IVF frequency of sperm incubated 6 hr with 10 mug/ml heparin (116 173 , 67%) was increased (P<0.05) above control levels (56 181 , 31%); however, 10 mug/ml CS-A (56 164 , 34%) was without effect (P>0.05). In contrast to previous reports, CS-A did not (P>0.05) induce AR in ejaculated (9.5-hr incubation) or epididymal sperm (22.5-hr incubation). Linear increases in fertilization frequency (40% to 81%; P=0.001) and AR (9% to 32%; P</=0.05) occurred with time of sperm exposure to heparin (15 sec to 6 hr) suggesting a direct effect of heparin on sperm. Glucose interfered with the effect of heparin on sperm. These data show heparin can prepare sperm for AR and fertilization in vitro and suggest that heparin-like material present in the female bovine reproductive tract may play a role in vivo in sperm capacitation and fertilization.

In vitro maturation and fertilization of bovine oocytes

Abstract Securing a plentiful and economical source of zygotes is central to capitalizing in the bovine on the many new genetic technologies currently available in both research and commercial settings. Using the fruits of the now mature A.I. industry and the many female germ cells wasted in numerous meat packing facilities, bovine zygotes can be produced entirely in vitro. Primary oocytes remoyed from antral follicles of abattoir-obtained ovaries can be induced to undergo in vitro the sequence of events found during in vivo periovulatory maturation of those oocytes in follicles selected to ovulate. Systems for in vitro maturation must ensure that the resulting oocyte has normally completed the first reduction division, is capable of undergoing normal fertilization and yields a zygote competent of developing to term after embryo transfer. Sperm from frozen-thawed semen can be capacitated in vitro with heparin to yield an in vitro fertilization system of high efficiency. By changing heparin dosage or sperm concentration, ejaculates can be adjusted to give comparable standards of fertilization. Ejaculates can also be characterized for precise timing of fertilization and developmental potential of zygotes produced. Our current knowledge concerning these aspects of gamete physiology as applied to the practical production of bovine zygotes in vitro will be discussed in this review.

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. When the number of oocytes from both peripheral and cortical follicles was combined, the yield of oocytes was approximately double that collected from 1 ovarian site alone. Oocytes from cortical follicles were smaller than those from the surface population, and the smaller cortical oocytes had a lower potential for both meiotic maturation and embryo development Only cortical oocytes with the largest diameters underwent IVM and subsequently developed to blastocysts at rates comparable to oocytes from peripheral follicles. As the diameter of the oocytes recovered from peripheral follicles increased, so did their developmental potential. When the stage of the estrous cycle was observed, it was found to have no effect on developmental potential. Finally, oocytes which extruded polar bodies at an earlier time during maturation were, on average, larger than those which extruded polar bodies later. The results serve a practical purpose in assisting selection of oocytes capable of developing into blastocysts and they give useful correlates of oocyte competencies based on knowledge of follicle source and oocyte stage.

Proteoglycan from bovine follicular fluid enhances an acrosome reaction in bovine spermatozoa

Abstract Bovine epididymal and ejaculated spermatozoa were incubated for 22 and 9.5 h respectively, in a chemically defined medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. Addition of bovine follicular proteoglycan or chondroitin sulfates ABC significantly increased the incidence of acrosome reaction. The stimulatory effects of the proteoglycan or chondroitin sulfates were negated by exposure to the enzyme chondroitinase ABC. Viability of spermatozoa was not affected by the various experimental treatments. Transmission electron microscopy of spermatozoa showed that vesiculation had occurred between the plasma and outer acrosomal membrane. These results suggest that proteoglycan present in follicular fluid at the time of ovulation may promote the acrosome reaction which precedes the ability of sperm to fertilize an ovum.

Glycosaminoglycans in bovine cumulus-oocyte complexes: morphology and chemistry.

Bovine cumulus-oocyte complexes from small (1-5 mm) follicles were cultured for 24 h in 0.25 ml minimum essential medium supplemented with 10% fetal bovine serum and 20 microCi [3H]glucosamine. Treatment groups consisted of supplementing the culture medium with no hormone (control), 0.5 IU/ml follicle-stimulating hormone (FSH) or 10 mM 8-Br-adenosine cyclic monophosphate (cAMP). After culture, the complexes were fixed for light and scanning electron microscopy. Electron photomicrographs revealed that complexes induced to expand with FSH or cAMP contained a copious glycosaminoglycan (GAG) matrix extending between and around the cumulus cells. Control complexes did not exhibit expansion or an extracellular matrix. The radiolabeled GAG material was isolated for chemical identification. Chemical analyses included: (1) electrophoresis of GAG material, (2) electrophoresis of GAG material after enzyme or nitrous acid treatment, (3) thin-layer chromatography of GAG hydrolysates. The results from electrophoresis showed that the radiolabeled GAG co- migrated with hyaluronic acid. The GAG material was resistant to chondroitinase ABC and nitrous acid degradation but was digested by hyaluronidase. Complexes treated with FSH and cAMP incorporated higher (P less than 0.1 and P less than 0.025 respectively) amounts of [3H]glucosamine into hyaluronic acid than control cultures. Thin-layer chromatography identified the primary amino sugar of the GAG to be glucosamine. These data collectively showed that the radioactive GAG produced by bovine cumulus-oocyte complexes was hyaluronic acid.

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.

Sexual dimorphism in IVM-IVF bovine embryos produced from X and Y chromosome-bearing spermatozoa sorted by high speed flow cytometry.

The objective of this study was to examine preimplantation development and sperm aster characteristics of bovine male and female embryos produced by using spermatozoa sorted for the X or Y chromosome. In vitro matured oocytes were inseminated at 24 h of maturation with sorted X or Y chromosome-bearing spermatozoa, using either fresh or frozen-thawed semen. Samples were taken from each sperm group 12 h post insemination (hpi), fixed, and immunostained for the microtubule cytoskeleton. Confocal microscopy enabled visualization of sperm aster formation and microtubule characteristics of each zygote during early fertilization. Cultured embryos were checked for cleavage at 30, 35, 40 and 45 hpi, embryo development was examined daily until Day 8 of culture. Blastocyst cell numbers were determined at the end of the experiments. Reanalysis of the sorted sperm cells for DNA content showed purity rates of 90.1 and 92.1% for X and Y chromosome-bearing spermatozoa, respectively. Reduced fertilization and development rates were observed when sorted spermatozoa were used compared with fresh and frozen-thawed spermatozoa. Penetration rates at 12 hpi were 39.5, 44.7, 55.9 and 79.0%, while blastocyst formation rates at Day 8 were 26.7, 26.5, 31.7 and 40.7% for X and Y chromosome-bearing spermatozoa, using fresh and frozen-thawed semen groups, respectively. Sperm aster size was larger in males than females, while the size of pronuclei and subjective grade of sperm aster quality showed no differences between sexes. In this study, a greater cleavage rate and sperm aster size in male embryos indicated a dimorphic pattern of development in male and female embryos during fertilization and first cleavage.

In vitro fertilization and development of bovine oocytes matured with commercially available follicle stimulating hormone

Abstract Immature bovine oocytes were cultured in a serum-free medium containing three sources of commercially available follicle stimulating hormone (FSH) or NIADDK-oFSH + oLH or NIADDK-oFSH preparations. Media also contained 1 μg/m1 estradiol-17β. In-vitro matured oocytes were fertilized in vitro and co-cultured with bovine oviductal epithelial tissue for blastocyst development. Frequencies of fertilization and development to the blastocyst stage in vitro did not differ (P>0.05) with commercially available FSH preparations and FSH standard (NIADDK-oFSH-17) in the presence or absence of luteinizing hormone standard (NIADDK-oLH-25). The results demonstrate that commercially available FSH preparations could be used successfully for in-vitro bovine oocyte maturation with subsequent production of bovine embryos.