Factors affecting embryo production in superovulated Bos taurus cattle.

Despite a long history of bovine superovulation research, significant commercial applications did not start until the early 1970s. For some 20 years thereafter, superovulation represented the primary tool for the production of cattle embryos. In the early 1990s, commercial invitro production (IVP) was initiated in cattle. Although ovum pick-up and IVP are now commercially practiced on a wide scale, superovulation and embryo recovery by flushing remain a widespread and very effective approach to the production of cattle embryos. This review covers both the history and the effects of multiple factors on superovulation in Bos taurus cattle. There are three general protocols for suitable pre-FSH programming of donors so that gonadotrophin-responsive follicles are available. Superovulation protocols vary widely based on the FSH source, the diluent used, the number and timing of FSH injections and the timing and utilisation of various prostaglandins, controlled internal progesterone releasing devices, gonadotrophin-releasing hormone, and other means of controlling follicular development and ovulation. The number of oocytes that can be stimulated to grow and ovulate within any given donor can be estimated by either ultrasound-guided sonography or by measuring concentrations of anti-Müllerian hormone in the blood. Animal-related factors that can influence the efficacy of superovulation include cattle breed, age, parity, genetics, lactational status and reproductive history. In addition, nutrition, stress, season, climate, weather and several semen factors are discussed.

A 100-Year Review: Reproductive technologies in dairy science.

Reproductive technology revolutionized dairy production during the past century. Artificial insemination was first successfully applied to cattle in the early 1900s. The next major developments involved semen extenders, invention of the electroejaculator, progeny testing, addition of antibiotics to semen during the 1930s and 1940s, and the major discovery of sperm cryopreservation with glycerol in 1949. The 1950s and 1960s were particularly productive with the development of protocols for the superovulation of cattle with both pregnant mare serum gonadotrophin/equine chorionic gonadotrophin and FSH, the first successful bovine embryo transfer, the discovery of sperm capacitation, the birth of rabbits after in vitro fertilization, and the development of insulated liquid nitrogen tanks. Improved semen extenders and the replacement of glass ampules with plastic semen straws followed. Some of the most noteworthy developments in the 1970s included the initial successes with in vitro culture of embryos, calves born after chromosomal sexing as embryos, embryo splitting resulting in the birth of twins, and development of computer-assisted semen analysis. The 1980s brought flow cytometric separation of X- and Y-bearing sperm, in vitro fertilization leading to the birth of live calves, clones produced by nuclear transfer from embryonic cells, and ovum pick-up via ultrasound-guided follicular aspiration. The 20th century ended with the birth of calves produced from AI with sexed semen, sheep and cattle clones produced by nuclear transfer from adult somatic cell nuclei, and the birth of transgenic cloned calves. The 21st century has seen the introduction of perhaps the most powerful biotechnology since the development of artificial insemination and cryopreservation. Quick, inexpensive genomic analysis via the use of single nucleotide polymorphism genotyping chips is revolutionizing the cattle breeding industry. Now, with the introduction of genome editing technology, the changes are becoming almost too rapid to fully digest.

Embryo production with sex-sorted semen in superovulated dairy heifers and cows.

The aim of this study was to examine the effect of sex-sorted semen on the number and quality of embryos recovered from superovulated heifers and cows on commercial dairy farm conditions in Finland. The data consist of 1487 commercial embryo collections performed on 633 and 854 animals of Holstein and Finnish Ayrshire breeds, respectively. Superovulation was induced by eight intramuscular injections of follicle-stimulating hormone, at 12-hour intervals over 4 days, involving declining doses beginning on 9 to 12 days after the onset of standing estrus. The donors were inseminated at 9 to 15-hour intervals beginning 12 hours after the onset of estrus with 2 + 2 (+1) doses of sex-sorted frozen-thawed semen (N = 218) into the uterine horns or with 1 + 1 (+1) doses of conventional frozen-thawed semen (N = 1269) into the uterine corpus. Most conventional semen (222 bulls) straws contained 15 million sperm (total number 30-45 million per donor). Sex-sorted semen (61 bulls) straws contained 2 million sperm (total number 8-14 million per donor). Mean number of transferable embryos in recoveries from cows bred with sex-sorted semen was 4.9, which is significantly lower than 9.1 transferable embryos recovered when using conventional semen (P ≤ 0.001). In heifers, no significant difference was detected between mean number of transferable embryos in recoveries using sex-sorted semen and conventional semen (6.1 and 7.2, respectively). The number of unfertilized ova was higher when using sex-sorted semen than when using conventional semen in heifers (P < 0.01) and in cows (P < 0.05), and the number of degenerated embryos in cows (P < 0.01), but not in heifers. It was concluded that the insemination protocol used seemed to be adequate for heifers. In superovulated cows, an optimal protocol for using sex-sorted semen remains to be found.

Synthetic media for culture, freezing and vitrification of bovine embryos.

Media designed for the recovery, holding and cryopreservation of bovine and equine embryos are available from several commercial sources. In years past, some of these media contained bovine serum, although inclusion of serum in embryo transfer media is now largely discontinued due to issues relating to storage and biosecurity. Currently, bovine serum albumin (BSA) is included in most commercially manufactured media intended for use in embryo transfer (ET). Although BSA poses less risk than serum for the transmission of infectious disease, its inclusion still entails risk of viral contamination. The present review briefly describes the various components of ET media and the development of efficacious ET media containing no products of animal origin. An evaluation of the efficacy of recovery, holding, slow controlled freezing and vitrification media in both research and commercial ET embryo transfer settings is also presented.

Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows.

To investigate why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, we compared these two approaches in the same Nelore cows (n=30) and assessed total embryo production and pregnancy rates. Without a specific schedule, all cows were subjected to ultrasound-guided ovum pick up (OPU)/in vitro production (IVP) and MOET, with intervals ranging from 15 to 45 d between procedures, respectively. To produce in vivo embryos, cows were superovulated and embryos were recovered nonsurgically from 1 to 3 times (1.4+/-0.6), whereas OPU/IVP was repeated from 1 to 5 times (3.2+/-1.2) in each donor cow during a 12-mo interval. Embryos obtained from both methods were transferred to crossbred heifers. On average, 25.6+/-15.3 immature oocytes were collected per OPU attempt. The average number of embryos produced by OPU/IVP (9.4+/-5.3) was higher (P<0.05) than the MOET method (6.7+/-3.7). However, pregnancy rates were lower (P<0.05) following transfer of IVP (33.5%) versus in vivo-derived embryos (41.5%) embryos. Embryonic losses between Days 30 and 60 and fetal sex ratio were similar (P>0.05) between in vivo and in vitro-derived embryos. We concluded that in Nelore cows, with an interval of 15 d between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional MOET.

Carbon-activated gas filtration during in vitro culture increased pregnancy rate following transfer of in vitro-produced bovine embryos.

Many environmental conditions for in vitro embryo production (IVP) systems for cattle have been relatively standardised, e.g. media composition, temperature, pH, water quality, and atmospheric composition. However, little attention has been paid to the quality of ambient laboratory air and the gas environment in incubators. Although a few studies have examined the effects of chemical air contamination on IVP of human embryos, there are no published accounts for domestic animal embryos. Therefore, this study investigated the effects of an intra-incubator carbon-activated air filtration system (CODA) during in vitro culture (IVC) on embryonic development and subsequent pregnancy rate of bovine embryos. Immature cumulus-oocyte-complexes (COCs) were obtained twice-weekly by ultrasonic-guided transvaginal oocyte aspiration. The COCs were matured in TCM199/FCS/LH/FSH, fertilized with frozen-thawed Percoll-separated semen, and subsequently cultured for 7 day in SOFaaBSA. Day 7 embryos were transferred either fresh or frozen/thawed. The experimental design was a 2 x 2 factorial; presumptive zygotes were placed either in a conventional CO(2)-O(2)-N(2) incubator (Control group) or in an identical CO(2)-O(2)-N(2) incubator with a CODA intra-incubator air purification unit (CODA group) for IVC. The embryo production rate at Day 7 was not affected by the CODA air purification unit (23.4 and 24.7% morulae and blastocysts per oocyte for control and CODA, respectively) nor was there any significant effect on embryo stage or quality. However, the pregnancy rate was improved (P=0.043) for both fresh (46.3% versus 41.0%) and frozen/thawed embryos (40.8% versus 35.6%). In conclusion, atmospheric purification by the CODA intra-incubator air purification unit significantly increased pregnancy rate following transfer of in vitro-produced bovine embryos.

Dissecting why superovulation and embryo transfer usually work on some farms but not on others.

Bovine embryo transfer is a well-established commercial industry that is often associated with veterinary practices. Practitioners offering embryo transfer services may possess a very high standard of technical expertise; however, success in the production of embryos and the impregnation of recipients cannot be achieved unless the cattle are healthy and maintained in a well-managed cattle operation. In addition to appropriate gonadotropin treatments of donor cattle, the use of highly fertile semen, known to have been properly stored and handled is required for success. Recipient cattle must be managed with the same attention to detail as donors. Traditionally, PGF has been used for the synchronization of recipients. However, PGF is limited in its effectiveness early and late in the bovine estrus cycle. Recipient estrus synchronization with progesterone releasing intravaginal inserts has been successful and high pregnancy rates have resulted following embryo transfer.

The Holstein cow in embryo transfer today as compared to 20 years ago.

Embryo transfer practice and results were examined over a 20-year period in Holstein cows and heifers within four commercial embryo transfer programs located in different areas of North America. Mean embryo production per collection decreased (P < 0.05) in one program over time, but not in the other three. Changes in the type of cows entering embryo transfer programs, the number of times they were superstimulated and changes in the brands of gonadotropins used for superstimulation all complicated the analysis of embryo production over time. Data reveal higher pregnancy rates (P < 0.001) following transfer of embryos into Holstein heifers than into lactating dairy cows. It is not clear whether pregnancy rates have decreased over time as a result of the change from surgical to non-surgical embryo transfer. In the two programs in which pregnancy rates were analyzed, there was a decrease (P < 0.001) when non-surgical transfers were adopted in one program, while no change occurred in the other. One of the biggest changes in all programs was that more than 50% of embryos recovered from donors are now frozen after collection, whereas the majority were transferred fresh 20 years ago.

Assisted reproductive technologies in cattle: a review.

Over a period of approximately thirty years, commercial bovine embryo transfer has become a large international business. The technology is well established, and more than 500,000 embryos are produced annually from superovulated cows world wide. Since bovine embryos with intact zonae pellucidae can be specified pathogen-free through washing procedures, thousands of frozen embryos are routinely sold and transferred between countries. Throughout the world, approximately 15% of bovine embryos are produced by in vitro technology. Polymerase chain reaction technology is currently being used for sexing embryos on a small scale, and it is likely that this technology will be used for 'embryo diagnostics' in the future. Semen sexing is an established technology and is likely to be used on a small scale in the near future, especially in in vitro embryo production systems. The cloning of adult cattle through nuclear transfer and the production of cloned, transgenic cattle has been technically achieved. However, this is an expensive and inefficient technology, which is being used primarily by the pharmaceutical industry. Benefits in agriculture are likely to be minimal in the near future.

Effect of recombinant bovine somatotropin on superovulatory response and recipient pregnancy rates in a commercial embryo transfer program.

Recombinant bovine somatotropin (rbST) has been shown to increase follicular growth in cattle and some studies have demonstrated an increase in superovulatory response for rbST-treated cows. Pregnancy rates have also been shown to increase when rbST was administered around the time of insemination or prior to embryo transfer. The application of rbST for the purpose of increasing superovulatory responses of donor cows and increasing pregnancy rates of recipient heifers was tested in a commercial embryo transfer program. In Experiment 1, embryo donor cows (n = 56) underwent three cycles of control superovulation (two before and one after weaning) and subsequently underwent up to four additional superovulations while being treated with either rbST (500 mg sustained-release rbST; Posilac, Monsanto, St. Louis, MO; n = 28) or excipient (control; n = 28) once every 14 days. In Experiment 2, lactating embryo donor cows (n = 37) underwent a control superovulation and then underwent a superovulation while lactating and being treated with either rbST (n = 16) or excipient (n = 21). In Experiment 3, embryo recipient heifers that were being implanted with either in vitro or in vivo produced embryos were treated with either rbST (n = 146) or excipient (n = 143) at the time of embryo transfer. Treatment of non-lactating (Experiment 1) or lactating (Experiment 2) donor cows with rbST during repeated superovulation did not affect the number of corpora lutea, the sum of transferable embryos, degenerate embryos, and unfertilized oocytes, or the number of transferable embryos. Treatment of recipient heifers with rbST (Experiment 3) did not affect pregnancy rates for either in vitro or in vivo produced embryos. We conclude that superovulatory response and pregnancy rates (respectively) are similar to control for rbST-treated cows undergoing repeated superovulations and rbST-treated recipient heifers treated at the time of embryo transfer.

Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment.

Techniques for sex determination of bovine embryos have evolved from karyotyping of older preimplantation embryos some 25 years ago to the current variety of widely used polymerase chain reaction (PCR) protocols. Although highly accurate, most PCR protocols for sex determination have included an electrophoresis step. The present work is a retrospective study utilizing a unique PCR protocol to sex bovine embryos without use of electrophoresis in a commercial embryo transfer program. Both in vivo and in vitro-derived embryos were produced by conventional techniques and biopsied between 7 and 8 days of age with a steel blade attached to a mechanical micromanipulator. Males constituted 49.0% of 3964 in vivo and 53.0% of 1181 in vitro-derived embryos subjected to PCR. Based on ultrasound fetal sexing and on calvings, the accuracy of sex determination was 98.7% for male embryos and 94.4% for females, with no samples producing an undetermined outcome. Pregnancy rates following transfer of biopsied Grade 1 embryos were lower than control, intact embryos as follows: 8, 6 and 16% points for in vivo, in vitro and in vivo frozen embryos, respectively. Pregnancy rates were similar for all stages of in vivo-derived embryos, whereas the pregnancy rate was significantly lower for in vitro-derived morulae compared to all stages of blastocysts. The sex ratio was significantly skewed in favor of females among in vitro-derived morulae, and in favor of males among in vitro expanded blastocysts. The sex ratio of in vivo expanded blastocysts was significantly skewed in favor of female embryos. No seasonal variation in either pregnancy rate or sex ratio was detected. There was no evidence that DNA contamination influenced the PCR assay during the duration of the study. The assay was sensitive to single blastomeres from male embryos, whereas it was not sensitive to Percoll-centrifuged or accessory sperm cells.

Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle.

The effects of a large number of factors on the pregnancy rates of fresh and frozen cattle embryos were examined over a period of years at several different locations. For fresh embryos, overall pregnancy rates were 68.3% (n=9023) and 77.1% (n=2650) at different locations and time periods. Frozen-thawed embryo pregnancy rates were 56.1% (n=3616) in The Netherlands and 58.4% (n=5297) and 68.7% (n=774) for two studies in the United States. Pregnancy rates of surgical versus nonsurgical transfers were very similar. There were no differences in the pregnancy rates of beef versus dairy embryos, but the pregnancy rate was higher in dairy and beef heifers and beef cows than in dairy cows. Although on-farm pregnancy rates in California were higher than in the northeast United States, there was no influence of season on pregnancy rate. Estrous asynchrony between plus and minus 24 h did not affect pregnancy rate for frozen-thawed or fresh embryos. Neither breed nor parity of recipients affected the influence of asynchrony on pregnancy rates. Embryo grade was a significant factor in pregnancy rate for both fresh and frozen-thawed embryos, but neither embryo stage nor age was a significant factor. Pregnancy rate was not affected by holding embryos after flushing for up to 3 h prior to freezing.

In vitro culture of bovine embryos in Ménézo's B2 medium with or without coculture and serum: the normalcy of pregnancies and calves resulting from transferred embryos.

The present study was designed to test the efficacy, as assessed by blastocyst production, of culturing slaughterhouse-derived bovine oocytes in Ménézo's B2 (B2) medium with or without serum and with or without buffalo rat cell (BRL) coculture. In addition, OPU-derived oocytes were cultured in B2-BRL coculture with or without serum for the first 72 h or TCM 199-BRL coculture with serum and the resulting embryos were transferred into recipients. Culture in B2 plus serum resulted in more blastocysts than B2 without serum, but both treatments produced far fewer blastocysts than did B2-BRL coculture. In addition, B2 with or without serum produced embryos of a lower stage of development, lower quality and fewer cells than did B2-BRL coculture. Serum restriction for the first 72 h of B2-BRL coculture did not result in a detectable difference in blastocyst production during 7 or 8 days of culture relative to coculture with no serum restriction. Embryos produced in B2-BRL coculture with serum restriction resulted in pregnancy rates, percentage of male fetuses, abortions, and congenital problems similar to those from B2 or TCM 199-BRL coculture with no serum restriction.

In-vitro production of cattle embryos: problems with pregnancies and parturition.

Using complex media containing serum and somatic cells, pregnancy rates following transfer of single, day 7 advanced-stage blastocysts approached 60%, while pregnancy rates of morulae or day 8 blastocysts were substantially lower. Pregnancies resulting from in-vitro-derived embryos were characterized by the following features: sex ratio skewed in favour of males; increased spontaneous abortion rate throughout gestation; reduced intensity of labour in recipients; and increases in birth weights, dystocias, calf mortality, and fetal abnormalities. In an attempt to improve the normality of pregnancies, a field trial was conducted within the commercial in-vitro programme at Em Tran. Following fertilization in vitro, equal numbers of zygotes were put into a Ménézo's B2-buffalo rat liver cell (B2-BRL) coculture system with (S+) or without (S-) 10% serum for the first 72 h of in-vitro culture. On day 4, all embryos were moved to fresh medium and cultured to day 7 in B2-BRL with serum. The efficiency of blastocyst production from oocytes (19.6 versus 17.8%) and the pregnancy rate at 60 days (47.8 versus 47.1%) did not differ between S+ and S-. Likewise, there was no difference between S+ and S- in the percentage of male calves (53.9 versus 54.3%), abortions (13.1 versus 11.9%), percentage of live calves (78.8 versus 81.4%), or congenital abnormalities (4.3 versus 3.3%).

Exposures to blood and body fluids among dental school-based dental health care workers.

A survey of United States dental schools was conducted to determine the annual incidence of reported percutaneous and mucosal exposures to blood and other body fluids among dental school-based dental health care workers (DHCW). A response rate of 51.9 percent provided information on 10,433 DHCW and 1.6 million student clinic and 169,836 school-based faculty practice patient visits. This response represents approximately half of all DHCW and student clinic visits in U.S. dental schools in AY 1996/1997. A total of 652 exposures were reported, of which 629 occurred in student clinics. Dental schools averaged twenty-three reported exposures per year, and the overall annual reported exposure rate in student clinics was 4.0/10,000 patient visits and 1.3/10,000 in faculty practice. Dental students accounted for 62.5 percent of all reported exposures, a rate of 106.3/1000 students per year. The exposure rate for dental students was significantly greater that any other category of DHCW. Expressed in terms of person years, an exposure rate of 0.17 was comparable to that reported for dental schools but considerably less than found in other dental care settings. A second survey directed to individual DHCW drew responses from only 8.3 percent of the 10,433 DHCW. Among these respondents, 31 percent of those acknowledging an exposure reported it. A judgment that the injury was not serious, the time necessary to report an exposure, and a belief that the patient was healthy were the primary reasons for not reporting. The results of this study provide dental schools with benchmarks for comparing their reported exposure experience and assessing programs intended to prevent and manage exposures.

Conception rates after artificial insemination or embryo transfer in lactating dairy cows during summer in Florida.

The objective was to compare conception rates to embryo transfer relative to AI, during summer heat stress, in lactating dairy cows. Holstein cows (n = 180; 50 to 120 d postpartum) were allocated randomly to 1 of 3 groups: artificial insemination (AI, n = 84), embryo transfer using either embryos collected from superovulated donors (ET-DON, n = 48), or embryos produced in vitro (ET-IVF, n = 48). Embryos from superovulated donors were frozen in 10% glycerol and were rehydrated in a 3-step procedure, in decreasing concentrations of glycerol in a sucrose medium before transfer. Embryos produced in vitro were frozen in 1.5 M ethylene glycol, thawed and transferred without rehydration. Blood samples were collected from AI and ET recipients on Days 0, 7 and 22 for measurement of progesterone in plasma. Conception rate was estimated for the three groups at Day 22 (progesterone > 1 ng/mL) and confirmed at Day 42 by palpation per rectum. Conception rate estimates at Day 22 did not differ among groups (AI, 60.7%; ET-DON, 60.4%; ET-IVF, 54.2%), but conception rates at Day 42 differed (AI, 21.4%; ET-DON, 35.4%; ET-IVF, 18.8%; AI versus ET: P > 0.10 and ET-DON versus ET-IVF: P < 0.05). In cows considered pregnant at 22 d but diagnosed open at 42 d, the interestrous intervals were 28.8 +/- 2.2, 35.2 +/- 3.5 and 31.6 +/- 2.9 d, respectively, for AI, ET-DON and ET-IVF groups. Transfer of embryos collected from nonheat-stressed superovulated donors significantly increased conception rates in heat stressed dairy cattle. However, transfer of IVF-derived embryos had no advantage over AI. Where appropriate mechanisms are in place to attenuate the effects of heat stress, embryo transfer using frozen-thawed donor embryos increases conception rates.

In vitro fertilization and in vitro culture of bovine embryos in the presence of noncytopathic bovine viral diarrhea virus.

In vitro embryo production has been used extensively in research and is now offered as a commercial service, yet the hazards of introducing specific infectious agents into in vitro embryo production systems have not been completely defined. The introduction of noncytopathic bovine viral diarrhea virus (BVDV) is a special concern. One objective of this study was to determine if noncytopathic BVDV-infected uterine tubal cells in IVF and IVC systems affected the rate of cleavage and development. An additional objective was to determine if either degenerated ova or embryos produced in the presence of the infected cells had virus associated with them after washing. Follicular oocytes (n = 645) collected from slaughterhouse ovaries were matured and fertilized in vitro, and presumptive zygotes were cultured for 7 d. Primary cultures of uterine tubal cells for use during IVF and IVC were divided into 2 groups. One-half of the cultures was infected with noncytopathic BVDV while the other half was not exposed to the virus. Approximately equal groups of mature oocytes were inseminated, and the presumptive zygotes were cultured with infected or noninfected uterine tubal cells. After 7 d in IVC, zona pellucida-intact (ZP-I) morulae and blastocysts and degenerated ova were washed and assayed for the presence of infectious virus. Infections of uterine tubal cells were not apparent and did not reduce rates of cleavage and development (P > 0.05; Chi-square test for heterogeneity). After washing, BVDV was isolated at a significantly higher rate from groups of virus-exposed degenerated ova (79%) than from individual virus-exposed morulae and blastocysts (37%; P = 0.0002; Mantel-Haenszel summary, Chi-square).

Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol.

Survival of IVF-derived bovine embryos of different ages and stages of development, produced in 2 different co-culture systems and frozen in 2 different cryoprotectants, was investigated. In vitro-derived bovine embryos (n = 5,525) were utilized to study survival following exposure to cryoprotectants and after freezing. Survival of the frozen embryos was based on blastocyst re-expansion 24 h and hatching 72 h after thawing. There was no difference in survival when embryos were exposed to either glycerol (Gly) or ethylene glycol (EG) for 10 or 40 min with the cryoprotectant diluted with or without freezing. In 2 of 3 experiments in which a comparison was possible, more blastocysts frozen in 1.4 M glycerol than in 1.5 M ethylene glycol survived. Addition of 0.25 M sucrose to 1.5 M ethylene glycol in the freezing solution did not improve embryo survival. More blastocysts frozen on Day 7 of in vitro culture survived than those frozen on Day 6 or Day 8. On Days 6, 7 and 8, embryos in the most advanced stage of development survived better than those at less advanced stages. Post-thaw survival did not differ for embryos produced in co-culture with Buffalo Rat Liver (BRL) cells with either Menezo B2 Medium or Tissue Culture Medium 199 and frozen in 1.4 M glycerol.

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts.

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.