Progress 10/01/99 to 09/30/04
In an attempt to better understand the underlying biological mechanisms controlling fertilization and embryonic development, a commercially available bull semen preparation product was utilized for in vitro embryo production experiments. Although an enhanced cleavage rate was obtained with use of this product, blastocyst formation rate was not different than controls. After segregation of Y chromosome bearing sperm from X chromosome bearing sperm using a multi-layered density gradient, embryos were produced in vitro and were subsequently assayed for embryonic sex. Embryonic sex ratios were not significantly altered from the expected 50:50 male:female sex ratio. In an effort to enhance control of the estrous cycle of postpartum cows to be used as embryo transfer recipients, dinoprost was compared with cloprostenol. These prostaglandin F2-alpha products were administered either intramuscularly or intralabially. Cows receiving cloprostenol tended to exhibit a higher rate
of estrus than cows receiving dinoprost, and intralabial administration of prostaglandin F2-alpha produced results similar to those obtained following intramuscular administration. It was concluded that intralabial administration should be adopted by producers as a means to avoid potential tissue damage in edible portions of the carcass, thus enhancing the quality of the nations' food supply. In an attempt to improve the pregnancy rate of recipient females receiving transferred embryos, two prostaglandin F2-alpha synthesis inhibitors were compared. Heifers receiving aspirin just prior to embryo transfer produced an 18% higher pregnancy rate than untreated heifers, while heifers receiving flunixin meglumine were intermediate. It is recommended that recipient females be given aspirin at the time of embryo transfer as a means to enhance embryo transfer pregnancy rate. A new study was initiated with the aim of increasing the number of ovarian follicles that will respond to exogenous
follicle stimulating hormone (FSH). Exogenous FSH is routinely administered to achieve superovulation of individual donor cows during the process of embryo transfer, but responses to this treatment are highly variable. A methodology that will yield consistent and predictable responses is sorely needed. Three different experimental treatments are being investigated for their effect on ovarian follicular dynamics and embryo production rate. This experiment is still in progress (data are still being collected); therefore; data analysis has not yet been performed. This work is being continued under project IOW06687.
Development of a protocol that will enhance the response of individual donor cows to superovulation with exogenous FSH will increase the application and utility of embryo transfer in cattle.
- Godke, R.A. and C.R. Youngs. 2004. Biotechnology: Embryo Technology for Cattle. In (W. Pond, Ed.) Encyclopedia of Animal Science (on-line). Marcel Dekker, New York, NY, DOI:10.1081/E-EAS 120019483, pp. 136-138.
- Pugh, M.L., M.B. Moreira, G.R. Gilbert and C.R. Youngs. 2004. Influence of prostaglandin F2a synthesis inhibitors on pregnancy rate of embryo transfer recipient heifers. Proc. 15th Int. Cong. Anim. Reprod. 2:399.
- Youngs, C.R., A.M. Powers-Meyer, M.G. Wonderlich and M.B. Moreira. 2004. The effect of cloprostenol or dinoprost, administered at two different sites, on the expression of estrus in postpartum dairy cattle. Proc. 15th Int. Cong. Anim. Reprod. 1:60.
Progress 01/01/03 to 12/31/03
A study was initiated to examine the effect of two different prostaglandin F2-alpha synthesis inhibitors (flunixin meglumine or aspirin) on the post-transfer pregnancy rate of recipients. A total of 383 embryo transfers was performed over a 19-month period. Embryos that had been cryopreserved in glycerol were purchased from a variety of sources, and frozen-thawed embryos were transferred by a single technician into naturally cycling recipients. Recipients received either 500 mg flunixin meglumine, 480 grains of aspirin or no treatment. Pregnancy rate was determined ultrasonographically at approximately Day 35 and again at Day 65 of gestation. Data were analyzed by chi-square analysis. There was no difference among treatments in Day 35 pregnancy rate that averaged 64%. However, heifers treated with aspirin tended (P<.07) to produce a higher Day 65 pregnancy rate (63%) than untreated control heifers (45%), with heifers treated with flunixin meglumine being intermediate
(57%). When aspirin and flunixin meglumine were combined and considered as a single treatment in a subsequent data analysis, Day 65 pregnancy rate (60%) was higher (P<.02) than controls. A second experiment was conducted using 231 mixed breed dairy cattle at a farm with a history of poor reproductive efficiency. Every two weeks, females who were at approximately 35-45 days postpartum underwent a routine reproductive exam. Any female who was deemed ready (by the farm manager) for breeding was allocated within breed to one of four treatments. Females possessing a corpus luteum (n=170), as verified by ultrasonography, received one of two PGF2-alpha treatments (500 mg cloprostenol or 30 mg dinoprost) administered either i.m. or intralabially (i.l.; within the vulvar lips). Cows were fitted with an estrus detection aid (Kamar patch) and were subsequently observed at least twice daily for signs of estrus. Treatments were considered successful if females exhibited estrus within 7 days of
treatment. Data were analyzed using chi-square. The incidence of estrus was not influenced by the site of PGF2-alpha injection (52% and 51% for i.m. and i.l., respectively). In contrast, the incidence of estrus tended (P<.07) to be higher in females treated with cloprostenol (58%, n=92) than in females treated with dinoprost (44%, n=78).
Results indicate that intralabial administration of prostaglandin F2-alpha is a viable alternative to traditional intramuscular administration. Results revealed that aspirin may be an effective blocker of prostaglandin F2a synthesis that could enhance pregnancy rate in recipients after embryo transfer.
- Youngs, C.R. 2003. Use of gonadotropin releasing hormone for controlled breeding of dairy cattle. Iowa Jersey Today 9(1):9-10.
Progress 01/01/02 to 12/31/02
Prostaglandin F2-alpha is a naturally occurring fatty acid hormone that is produced by the endometrium of the cow. This hormone is released from the endometrium near the end of the estrous cycle in instances when the preimplantation embryo either is not present or does not emit a sufficiently strong signal for maternal recognition of pregnancy. Prostaglandin F2-alpha induces luteolysis, which enables the cow to subsequently exhibit estrus and ovulate. Poor reproductive efficiency is often observed in high-producing dairy cattle during the postpartum period. One method to achieve an increase in the number of breeding opportunities of these females is to induce estrus with exogenous prostaglandin F2-alpha. A study was initiated in postpartum dairy cattle to investigate the use of two different commercially available prostaglandin F2-alpha products (dinoprost, cloprostenol) using two different routes of administration (intramuscular, intravulvar). Response to the
exogenous prostaglandin F2-alpha treatments is being monitored with the aid of an electronic system for detection of estrus called HeatWatch. The HeatWatch system continuously monitors estrous activity in cows and records the number, duration, and timing of mounting activity. Gaining precise knowledge on cows' response to exogenous prostaglandin F2-alpha treatment will enable subsequent studies on fertilization and embryonic development to be conducted. This information will also be highly useful for the commercial bovine embryo transfer industry. Preliminary estrous data have been collected and are being analyzed, but data on pregnancy rates and calving rates are not yet available for analysis.
By using the HeatWatch system for detection of estrus, it will enable researchers to more closely pinpoint the timing of events related to fertilization and early preimplantation embryonic development in the cow. Gaining a better understanding of underlying biological mechanisms of fertilization and embryonic development is crucial to enable enhanced success rates of embryo transfer and associated technologies such as in vitro fertilization.
- Godke RA, M Sansinena and CR Youngs. 2002. Assisted reproductive technologies and embryo culture methods for farm animals. In (C.A. Pinkert, Ed.) Transgenic Animal Technology: A Laboratory Handbook, 2nd Ed. Academic Press, San Diego, CA, pp. 513-568.
- Youngs C. 2002. 2002 IETS foundation student research competition. Embryo Transfer Newsletter 20(1):4.
- Youngs CR. 2002. The magnificent seven hormones controlling reproduction in the cow (Part II). Iowa Jersey Today 8(1):5-6.
- Youngs CR. 2002. Synchronization of estrus in dairy cattle using prostaglandin F2-alpha (Part I). Iowa Jersey Today 8(2):10.
- Youngs CR. 2002. Synchronization of estrus in dairy cattle using prostaglandin F2-alpha (Part II). Iowa Jersey Today 8(3):9-10.
Progress 01/01/01 to 12/31/01
Previous reports have indicated that genetic sex of a mammalian preimplantation embryo influences the rate of embryonic development. To better study reasons for differences in embryonic developmental rate, it would be useful to produce embryos of a known genetic sex so that embryonic sex is not confounded with treatment effects. A multi-layer density gradient, prepared using a commercial product (BoviPureTM), was used in an attempt to separate X and Y chromosome-bearing bovine sperm. Separated sperm were subsequently used for in vitro production of bovine embryos. Cleavage rate differed (P<.01) between BoviPureTM and control groups (77.2 vs. 64.5%, respectively). However, no difference (P>.05) was found in rate of blastocyst formation (19.7%). There was no effect (P>.05) of treatment on embryonic sex ratio between oocytes fertilized with control sperm or sperm separated through a BoviPureTM density gradient on either Day 2 (50% male for BoviPureTM versus 53% male for
control) or Day 8 (74% male for BoviPureTM versus 67% male for control). In a related study, polymorphisms in the amelogenin gene are being used as the basis for development of a simplified embryo sexing assay. Using polymerase chain reaction, bands of DNA specific to the X- and Y-chromosomes are being amplified. Preliminary results indicate that bovine embryonic sex may be determined approximately 50% of the time. Further refinements to the protocol are being investigated in order to improve the efficiency of the technique.
The ability to understand, control, and manipulate early preimplantation embryonic development will profoundly impact reproductive efficiency in domestic mammalian livestock species. Results of this research indicate that segregation of sperm using density gradients can enhance the in vitro production of bovine embryos. This could be of great interest and benefit to dairy and beef cattle producers.
- Youngs CR. 2001. Factors influencing the success of embryo transfer in the pig. Theriogenology 56:1311-1320.
- Sieren KR. 2001. The Use of BoviPureTM for the Separation of X and Y Chromosome-Bearing Bovine Sperm Cells. M.S. Thesis, Iowa State University, 61 pages.
- Youngs CR, Ed. 2001. Proc. of the International Conference Commemorating the 50th Anniversary of the First Successful Embryo Transfer in the Pig. Theriogenology 56(8):1285-1369.
- Dikeman MA, DR Strohbehn and CR Youngs. 2001. Development of a web-based scheduling program for synchronization of estrus in cattle. J. Anim. Sci. 79(Suppl.2):15.
- Funk DJ and CR Youngs. 2001. Embryo transfer, in vitro fertilization, cloning and transgenic production in farm animals: A review. Proc. of the Student American Veterinary Medical Association Symposium, March 8-10, Ames, IA, p. 89
- Pugh ML, KR Sieren, LL Timms and CR Youngs. 2001. Use of a vaginal mucus electrical resistance probe to alter insemination time and calf gender distribution in dairy heifers. J. Dairy Sci. 84(Suppl. 1):64.
- Sieren KR and CR Youngs. 2001. Evaluation of BoviPureTM for the in vitro production of bovine embryos. Theriogenology 55:438.
- Sieren KR and CR Youngs. 2001. Assessment of BoviPureTM for the in vitro production of bovine embryos. Beef Research Report, Iowa State University, A.S. Leaflet R1768, pp. 142-144.
- Youngs CR. 2001. Cattle breeding in the 21st century. Proc. of the 30th Annual Cornbelt Cow-Calf Conference, February 24, Ottumwa, IA, pp. 1-9.
- Youngs CR. 2001. Sheep and goat reproduction. Proc. of the Student American Veterinary Medical Association Symposium, March 8-10, Ames, IA, pp. 122-128.
- Youngs CR. 2001. A new millennium for the Jersey breed. Iowa Jersey Today 7(2):5.
- Youngs CR. 2001. The Magnificent Seven... hormones controlling reproduction in the cow (Part I). Iowa Jersey Today 7(3):6.
- Youngs CR. 2001. Use of a vaginal mucus electrical resistance probe as a breeding tool for dairy cattle. Swiss Valley Dairyman, November issue, pp. 6-7.
- Pugh ML, M Pence, JN Caamano, S Robbe, LL Timms, JU Thomson and CR Youngs. 2001. Use of a vaginal mucus electrical resistance probe to alter insemination time and calf gender distribution in beef heifers. J. Anim. Sci. 79(Suppl. 2):66-67.
Progress 01/01/00 to 12/31/00
Objective 1. The in vitro production of bovine embryos is a multi-step process involving the in vitro maturation of oocytes, in vitro capacitation of sperm, in vitro fertilization of matured oocytes, and in vitro culture of resultant embryos. One aspect of bovine in vitro embryo production that varies considerably from laboratory to laboratory is the sperm preparation procedures. Recently, a product formulated specifically for the separation and purification of sperm from bovine semen (BoviPure) was introduced. An experiment was initiated to evaluate the use of BoviPure for in vitro production of bovine embryos. In vitro matured bovine oocytes were fertilized with semen prepared in BoviPure or control medium (modified Brackett-Oliphant medium). After a 6-hour insemination period, presumptive zygotes were freed of cumulus cells and placed into culture. Cleavage rate and blastocyst formation rate of embryos were assessed after 2 and 8 days of culture, respectively. Data
from six replicates were analyzed by chi-square analysis. Cleavage rate was higher (P<.01) in oocytes fertilized with sperm prepared with BoviPure, but blastocyst production rate was similar between groups.
The ability to understand, control, and manipulate early preimplantation embryonic development will profoundly impact reproductive efficiency in domestic mammalian livestock species. Results of this research will enhance the ability to produce bovine embryos using in vitro methods.
- Kobrinovitch FN, VS Kot, L Gorbunov, CR Youngs and ND Bezugly. 1999. Creation of an embryo cryobank for cattle of the Grey Ukrainian breed. Cryobiology 39:349-350.
- Morozova I, L Gorbunov and CR Youngs. 1999. Vitrification properties of glycerol-sucrose solutions at different cooling/thawing speeds. Proc. 36th Ann. Mtg. Soc for Cryobiology, July 12-15, Marseille, France, abstr. 204.
- Youngs CR, G Zhernoklyov, A Medvedovsky, N Godienko and N Bezugly. 1999. Osmotic properties of the pig late (expanded, hatching and hatched) blastocysts. Proc. 36th Ann. Mtg. Soc for Cryobiology, July 12-15, Marseille, France, abstr. 202.
- Perez O, R Richard III, HL Green, CR Youngs and RA Godke. 2000. Ultrasound-guided transvaginal oocyte recovery from FSH-treated post-partum beef cows. Theriogenology 53:364.
- Youngs CR. 2000. IETS Foundation competition award winners for 2000. Embryo Transfer Newsletter 18(2):18.
Progress 01/01/99 to 12/31/99
IDENTIFICATION OF MARKER SYSTEMS FOR OOCYTE QUALITY AND EMBRYONIC DEVELOPMENT. Developmental potential of oocytes recovered from postpartum cows was investigated. Excellent quality oocytes from postpartum cows developed into blastocysts at a rate (15.4%) not different than that for control oocytes (24.8%) obtained from abattoir ovaries. A significant effect of donor female on developmental competence was observed. REFINE METHODS FOR SHORT- AND LONG-TERM MAINTENANCE OF EMBRYOS. Oxygen free radicals are believed to adversely impact in vitro embryonic development. One of the potential mechanisms of cell defense to avoid damage induced by oxygen free radicals is via the compound glutathione. Maturation of bovine oocytes with 5 micromolar cysteamine enhanced blastocyst formation rate, presumably by maintaining intracellular glutathione concentration. Porcine embryos have been difficult to cryopreserve using conventional freezing methods. To better understand the underlying
biological constraints to porcine embryo cryopreservation, the osmotic properties of peri-hatching blastocyst stage porcine embryos was studied. The relative volume of osmotically active water increases throughout embryonic development, suggesting that osmotically active soluble substances are utilized for membrane synthesis.
The ability to understand and manipulate early embryo development will profoundly impact reproductive efficiency in domestic mammalian livestock species. Results of this research will enhance the in vitro production of bovine embryos and will lead to a better understanding of factors hindering the cryopreservation of porcine embryos.
- Kobrinovitch FN, Kot VS, Gorbunov L, Youngs CR, and Bezugly ND. 1999. Creation of an embryo cryobank for cattle of the Grey Ukrainian breed. Cryobiology 39(4):(in press).
- Morozova I, Gorbunov L and Youngs CR. 1999. Vitrification properties of glycerol-sucrose solutions at different cooling/thawing speeds. Cryobiology 39(4):(in press).
- Youngs CR, Zhernoklyov G, Medvedovsky A, Godienko N, and Bezugly N. 1999. Osmotic properties of the pig late (expanded, hatching and hatched) blastocysts. Cryobiology 39(4):(in press).
- Geshi M, Youngs CR, and Nagai T. 1999. Addition of cysteamine to a serum-free maturation medium enhances in vitro development of IVM-IVF bovine oocytes. J. Mamm. Ova Res. 16:135-140.
- Pugh ML, Pence M, Caamano JN, Robbe S, Timms LL, Thomson JU, BreDahl R, and Youngs CR. 1999. The use of a vaginal conductivity probe to influence calf sex ratio via altered insemination time. Beef Research Report, Iowa State University, A.S. Leaflet R1652, pp. 112-120.
- Pence M, Pugh ML, Robbe S, Timms L, Caamano JN, Thomson JU, Youngs CR, and BreDahl R. 1999. Comparison of various methods of estrus detection in synchronized virgin beef heifers. Beef Research Report, Iowa State University, A.S. Leaflet R1655, pp. 132-133.
- Pugh ML, Timms, and Youngs CR. 1999. Preliminary report on the use of a vaginal conductivity probe to alter calf sex ratio. Dairy Report, Iowa State University, DSL-171, pp. 70-71.
- Seidel GE Jr, Youngs CR, and Godke RA. 1999. Assisted Reproductive Technologies in Farm Animals: A Historical Perspective. Educational Tutorial Slide Set, International Embryo Transfer Society Foundation 2:1-75.
- Kuhholzer B, Pugh ML, Youngs CR and Overstrom EW. 1999. In vitro maturation of transported pig oocytes in a defined protein-free medium. Theriogenology 51:382.
Progress 01/01/98 to 12/31/98
IMPACTS OF RESEARCH. The ability to understand, control, and manipulate early embryonic development will profoundly impact reproductive efficiency in mammalian livestock species. Results of this research will enable substantial gains in the in vitro production of bovine embryos, novel contributions of postpartum cows to genetic improvement programs, and alternative methods for selective genetic modification of animals. IDENTIFY MARKERS FOR OOCYTE QUALITY AND EMBRYONIC DEVELOPMENT. Developmental potential of oocytes recovered via transvaginal ultrasound-guided oocyte retrieval from postpartum dairy cows was examined. Tremendous animal to animal variation in developmental competence of the oocytes was observed (range of 2 to 22% blastocyst formation rate). Efforts are underway to identify reasons for these developmental differences. REFINE METHODS FOR SHORT-TERM MAINTENANCE OF EMBRYOS. Oxygen free radicals are believed to adversely impact in vitro embryonic development.
Enhanced development was obtained by reducing the oxygen level of the incubation atmosphere from 20% to 5% and by the inclusion of 20 micromolar beta-mercaptoethanol. Cell number of resultant blastocysts was greater for embryos cultivated in the low oxygen atmosphere. DEVELOP TECHNIQUES FOR PRODUCTION OF GENETICALLY MODIFIED ANIMALS. Primordial germ cells may be useful for the production of genetically modified animals. The genital ridge was isolated from day 24 to 27 porcine conceptuses, and various enzymatic and mechanical methods for isolation of primordial germ cells were employed. Attempts to isolate primordial germ cells using a flow cytometer were less fruitful than traditional isolation methods.
- Caamano, J.N., Ryoo, Z.Y. and Youngs, C.R. 1998. Promotion of development of bovine embryos produced in vitro by addition of cysteine and b-mercaptoethanol to a chemically defined culture system. J. Dairy Sci. 81:369-374.
- Wilson, M.E., Biensen, N.J., Youngs, C.R. and Ford, S.P. 1998. Development of Meishan and Yorkshire littermate conceptuses in either Meishan or Yorkshire uterine environment to day 90 of gestation and to term. Biol. Reprod. 58:905-910.
- Caamano, J.N., Pugh, M.L., Timms, L.L. and Youngs, C.R. 1997. Preliminary report on the developmental potential of oocytes recovered from postpartum dairy cows. Dairy Report, Iowa State University, DSL128, pp. 48-50.
- Pugh, M.L., Maxwell, D.R. and Youngs, C.R. 1998. The efficacy of an 8-day MGA/PGF2a treatment for synchronization of estrus in crossbred beef heifers. Iowa State Univ. McNay Memorial Res Demonstr Farm 1997 Ann. Prog. Rpt., pp. 28-34.
- Pugh, M.L., Timms, L.L. and Youngs, C.R. 1998. Preliminary report on the use of a vaginal conductivity probe to alter calf sex ratio. Dairy Report, Iowa State University, DSL 171, (in press).
- Caamano, J.N., Pugh, M.L., Rowson, A.D. and Youngs, C.R. 1998. Effects of oxygen tension and b-mercaptoethanol on development of in vitro produced bovine embryos. Theriogenology 49:196.
Progress 01/01/97 to 12/31/97
IDENTIFY MARKERS FOR OOCYTE QUALITY AND EMBRYONIC DEVELOPMENT. Reproductive performance of cows during the postpartum period is often poor. The specific reasons for the observed infertility have not been elucidated. A study was initiated to examine developmental potential of oocytes recovered via transvaginal ultrasound-guided oocyte retrieval from postpartum dairy cows. Harvested oocytes were matured, fertilized and cultured in vitro. At least one blastocyst was produced from each cow, but tremendous animal to animal variation in developmental competence of the oocytes was observed. Efforts are underway to identify reasons for these developmental differences. REFINE METHODS FOR SHORT-TERM MAINTENANCE OF EMBRYOS. Taurine is a compound which is believed to have antioxidant activity. Four- to eight-cell bovine embryos produced in vitro were randomly allocated to one of four different doses of taurine (0. 0.1, 1, and 10 mM) in medium 199. Embryos were cultured for 6
days, and blastocyst formation rate was determined. Results revealed that the addition of 1 mM taurine enhanced blastocyst formation rate. DEVELOP TECHNIQUES FOR PRODUCTION OF GENETICALLY MODIFIED ANIMALS. Retroviruses are very efficient in transferring their genes to the host cells which they infect. The zona pellucida may serve as a barrier to retroviral-mediated gene transfer, so a study was initiated to assess the developmental potential of in vitro produced bovine embryos whose zona pellucidae had been removed by digestion with pronase. Development to the blastocyst stage was not affected by removal of the zona pellucida; however, cell number in the resultant blastocysts tended (P<.09) to be less for zona-free (67 cells) than for zona-intact embryos (80 cells).
- CAAMANO, J.N., RYOO, Z.Y. and YOUNGS, C.R. 1997. Cysteine and beta-mercaptoethanol promote development of in vitro produced bovine embryos in a chemically defined culture system. J. Dairy Sci. (In
- WILSON, M.E., BIENSEN, N.J., YOUNGS, C.R. and FORD, S.P. 1997. Development of Meishan and Yorkshire littermate conceptuses in either a Meishan or Yorkshire uterine environment to day 90 of gestation and to term. Biol. Reprod. (In press).
- CAAMANO, J.N., PUGH, M.L., TIMMS, L.L. and YOUNGS, C.R. 1997. Preliminary report on the developmental potential of oocytes recovered from postpartum dairy cows. Dairy Research Report, Iowa
- CAAMANO, J.N. and YOUNGS, C.R. 1997. Effect of taurine on development of in vitro produced bovine embryos. J. Anim. Sci. 75(Suppl. 1):86.
- GESHI, M., NAGAI, T. and YOUNGS, C.R. 1997. Effect of zona pellucida removal on in vitro development of 2- to 4-cell bovine embryos obtained via IVM-IVF. Theriogenology 47:306.
- MCKINNEY, S.L., YOUNGS, C.R., PROUGH, S.G., BLACKWELL, J., GEHRING, P.J. and THIBODEAUX, J.K. 1997. Quantitative measurements of pronuclei within human zygotes. Theriogenology 47:195.
Progress 01/01/96 to 12/30/96
IDENTIFY MARKERS FOR OOCYTE QUALITY AND EMBRYONIC DEVELOPMENT. Oocytes were harvested from a single Holstein cow using transvaginal ultrasound-guided oocyte retrieval. Harvested oocytes were matured, and all cumulus cells were removed. The D-loop region of the mitochondrial (mt) genome was amplified via the polymerase chain reaction (PCR). The mtDNA sequence was determined using an automated DNA sequencer. Results revealed that 45 of 47 oocytes (96%) had identical mtDNA sequence. The remaining two oocytes had one or two sites where their genetic make-up differed from their counterparts. Three of the 635 sites examined were different from the reference sequence. REFINE METHODS FOR SHORT-TERM MAINTENANCE OF EMBRYOS. A 2 x 3 factorial treatment arrangement was used to evaluate the effect of 0.63 or 6.9 uM cysteine and 0, 10 or 100 uM beta-mercaptoethanol (BME) on development of bovine embryos in a chemically defined medium. In vitro produced 6- to 8-cell embryos were
cultured for 7 days. Blastocyst formation rate was enhanced by BME (P<0.001) and cysteine (P<0.01), but no interaction existed. Blastocyst formation did not differ (P>0.11) between 10 uM or 100 uM BME but was higher than for embryos cultured without BME. Blastocyst cell number was enhanced by BME (P<0.01) but was not affected by cysteine (P>0.74). These findings provide evidence that BME and cysteine promote increased embryo development and that BME increases cell number of bovine in vitro produced embryos.
- Caamano, J.N., Ryoo, Z.Y., Thomas J.A., Youngs, C.R. 1996. B-mercaptoethanol enhances blastocyst formation rate of bovine in vitro matured/in vitro fertilized embryos. Biol Reprod 55:1179-1184
- Rivera, R.M., Youngs, C.R., Ford S.P. 1996. Comparison of number of inner cell mass and trophectoderm cells of preimplantation Meishan and Yorkshire pig embryos at similar developmental stages. J Reprod Fertil 106:111-116
- Kaminski, M.A., Ford, S.P., Youngs, C.R., Conley, A.J. 1996. Lack of effect of sex on pig embryonic development in vivo. J Reprod Fertil 106:107-110
- Geshi, M., Youngs, C.R., Nagai, T. 1996. Addition of cysteamine to a serum-free maturation medium enhances in vitro development of IVM-IVF bovine oocytes. Proc 8th AAAP Anim Sci Congr, Makahari, Chiba, Japan, October 13-18, 2:484-485
- Caamano, J.N., Ryoo, Z.Y., Youngs, C.R. 1996. Beneficial effects of cysteine and B-mercaptoethanol on development of bovine IVM/IVF embryos in cell-free, serum-free culture system. Theriogenology 45:196
- Geshi, M., Youngs, C.R. 1996. Effect of neomycin concentration on development of bovine preimplantation embryos derived from in vitro maturation and fertilization. J Anim Sci 74(Suppl 1):70
- Youngs, C.R. 1996. Use of embryo transfer to identify potential strategy for increased litter size in the pig. Proc Symp Anim Biotech, March 12, Tsukuba, Japan, pp 27-34.
Progress 01/01/95 to 12/30/95
IDENTIFY MARKERS FOR OOCYTE QUALITY AND EMBRYONIC DEVELOPMENT. Mitochondrial DNA(mtDNA) of individual bovine oocytes was isolated, amplified and sequenced to examine genetic variation in mtDNA sequence within oocytes of a given cow and among oocytes of different cows. All eight oocytes examined had identical mtDNA D-loop sequence in seven of eight cows. Oocytes from the eighth cow possessed identical sequence in seven of the eight oocytes. When comparing mtDNA sequence among cows, nucleotide variation was detected at 12 of the 635 sites examined. REFINE METHODS FOR SHORT-TERM MAINTENANCE OF EMBRYOS. The effect of B-Mercaptoethanol (0 or 100 uM) and fetal bovine serum (0 or 10%) on in vitro development of in vitro matured, in vitro fertilized bovine embryos was investigated. Embryos were cultured in medium 199 for 6 days at 39 degrees C in an atmosphere of 5% CO2. Fewer (P<.01) 6- to 8-cell embryos formed blastocysts compared with embryos initially containing 8-16
cells. Both FBS and BME significantly affected blastocyst formation, but only BME increased cell number of the resultant blastocysts. DEVELOP TECHNIQUES FOR PRODUCTION OF GENETICALLY MODIFIED ANIMALS. Intact blastocysts or inner cell masses isolated via calcium ionophore treatment were seeded onto STO feeder layers cultivated in modified DMEM. The efficiency of isolation and establishment of sheep embryo-derived cell colonies and cell lines was higher (P<.05) for the calcium ionophore treatment than for the intact blastocysts.
- Rivera, R.M., C.R. Youngs and S.P. Ford. 1995. A comparison of inner cell mass and trophectoderm cell numbers of preimplantation Meishan and Yorkshire pig embryos at similar developmental stages (submitted to Journal of Reproduction and Fer
- Kaminski, M.A., S.P. Ford, C.R. Youngs and A.J. Conley. 1995. Lack of effect of sex on porcine embryonic development (submitted to Journal of Reproduction and Fertility).
- Dunn, A.K., T.H. Brandes, R.K. Smith and C.R. Youngs. 1995. Investigation of genetic variation in mitochondrial DNA sequence of bovine oocytes. Beef Research Report, Iowa State University, A.S. Leaflet R1225, pp. 44-47
- Dunn, A.K., C.R. Looney, B.R. Lindsay and C.R. Youngs. 1995. Genetic variation in mitochondrial DNA sequence of bovine oocytes from a single dairy cow. Dairy Research Report, Iowa State University, DSL-30, pp. 59-61
- Godke, R.A., D.M. Barry, M. Meintjes and C.R. Youngs. 1995. New assisted reproductive technologies for domestic goats. Proc., Goat Production & Marketing Opportunities in the South, August 19, Baton Rouge, LA, pp. 23-36
- Bezugly, N., C. Youngs, A. Medvedovsky and E. Valigura. 1995. Osmotic composition of pig ova and embryos. Proc. 11th Scientific Meeting of the European Embryo Transfer Association, p. 128
- Biensen, N.J., M.E. Wilson, C.R. Youngs and S.P. Ford. 1995. Differential vascu.
Progress 01/01/94 to 12/30/94
IN VITRO OOCYTE MATURATION, FERTILIZATION, AND CULTURE. The effect of osmotic pressure of culture medium on in vitro development of IVM/IVF bovine oocytes was investigated. Presumptive fertilized oocytes were cultured in CR1aa medium containing various concentrations of NaCl (50 to 150 mM). Best results were obtained in slightly hypotonic osmotic pressures (235, 256 and 271 mOsm). Other osmotic pressures inhibited cleavage and/or in vitro development. EMBRYO STORAGE. The objective of this study was to obtain preliminary data on the phospholipid (PL), cholesterol (CHOL), triacylglycerol (TAG), and fatty acid (FA) composition of porcine preimplantation embryos. PL content of Day 10 and 11 embryos was 0.31 and 0.54 ug, respectively. CHOL content was 5.38 (Day 10) and 7.09 (Day 11) ug. TAG content was 1.13 (Day 10) and 1.41 (Day 11) ug. Oleic acid comprised 41% of the embryonic FA content. These data are the first on biochemical composition of porcine blastocysts and may
provide insights into cryopreservation protocols. GENE TRANSFER. Factors that may influence isolation of sheep embryonic stem (ES) cells were investigated. Intact blastocysts (IB) or inner cell masses isolated via either immunosurgery (IS) or calcium ionophore treatment (CIT) were studied. IS may be better than CIT or IB and Day 8 may be more suitable than Day 9 for isolation of sheep ES-like colonies. Insulin was highly effective in promoting formation of ES-like colonies, and EGF yielded good but slightly poorer results.
- YOUNGS, C.R., CHRISTENSON, L.K. AND FORD, S.P. 1994. Investigations into the control of litter size in swine: III. A reciprocal embryo transfer study of early conceptus development. J. Anim. Sci. 72:725-731.
- WEBER, P.K. AND YOUNGS, C.R. 1994. Investigation of cryoprotectant toxicity to porcine embryos. Theriogenology 41:1291-1298.
- TSUCHIYA, Y., BRANDES, T.L. AND YOUNGS, C.R. 1994. Effect of osmotic pressure on in vitro culture of IVM/IVF bovine oocytes. Proc. N.Z. Embryo Transfer Workshop, January 14-15, Hamilton, NZ, pp. 88-89.
- FORD, S.P., ET AL. 1994. Inhibitory effects of the Meishan uterus on growth rate and estradiol-17B (E2) secretion of day 30 conceptuses. Biol. Reprod. 50(Suppl. 1):175.
- RIVERA, R.M., CHRISTENSON, L.K., YOUNGS, C.R. AND FORD, S.P. 1994. Competitive survival, growth rate and estrogen secretory activity of Meishan and Yorkshire fetuses. J. Anim. Sci. 72(Suppl. 1):78.
- RIVERA, R.M., YOUNGS, C.R., CHRISTENSON, L.K. AND FORD, S.P. 1994. Inner cell mass (ICM) and trophectoderm (TE) cell numbers in Meishan and Yorkshire pig blastocysts. J. Anim. Sci. 72(Suppl. 2):74.
- TSUCHIYA, Y., RAASCH, G.A., BRANDES, T.L., MIZOSHITA, K. AND YOUNGS, C.R. 1994. Isolation of ICM-derived cell colonies from sheep blastocysts. Theriogenology 41:321.
Progress 01/01/93 to 12/30/93
IN VITRO OOCYTE MATURATION, FERTILIZATION, AND CULTURE. The effect of antibioticand serum inactivation on development of bovine oocytes was investigated. Media contained fetal bovine serum (FBS) that either was or was not heat-inactivated as well as one of three different antibiotics: 50 ug/ml gentamicin, 1% penicillin-streptomycin, or 1% antibiotic-antimycotic (pen-strep + amphotericin B). Neither maturation or cleavage rates was affected by treatments. However, gentamicin inhibited (P<.05) blastocyst formation. EMBRYO STORAGE. Five different concentrations (10, 20, 30, 40, or 50%) of each of three different cryoprotectants (ethylene glycol (EG); propylene glycol (PG); glycerol (GLYC)) were examined for their toxicity to pig embryos. Embryos at the compact morula through hatched blastocysts stages were placed into cryoprotectant treatments, followed by cryoprotectant removal and subsequent in vitro culture. Embryos exposed to GLYC and PG exhibited poorer (P<.05)
development than those exposed to EG, especially at the 40 and 50% concentrations. GENE TRANSFER. Two methods for inner cell mass (ICM) isolation from porcine blastocysts, immunosurgery or calcium ionophore treatment, were compared. ICM isolation rate was higher (P<.01) for calcium ionophore than for immunosurgery. ICM seeded onto STO feeder layers yielded ICM colonies at a rate of 57% or 0%, respectively, for ionophore and immunosurgery. Use of calcium ionophore may enhance ability to isolate porcine ES cells.
Progress 01/01/92 to 12/30/92
In this study,the association between graded dietary histidine(his) levels,plasma his and intramuscular carn were evaluated in eight mature Quarter Horse geldings. The horses were used in a 4x4 Latin square design. Four diets containing his levels of 0.2%,0.36%,0.45%,and 0.56% were fed two times per dayn at a rate to maintain constant body weight. Prior to and at the end of each five week period,jugular blood samples and middle gluteal muscle samples were obtained 3 hours after a meal. Plasma samples were analyzed for his,urea-N,hemoglobin and total protein. Muscle samples were analyzed for carnosine(carn),his and B-alanine (B-ala). Plasma urea-N,hemoglobin and total protein were not (>.05) altered by diet. Plasma his increased (P<,0001) with increasing dietary his with an inflection point occurring at 0.56% his. Middle gluteal free carn was not (P>,05) affected by diet, averaging 94.41 +/- 3.45,96.71 +/- 3.98,99.55 +/- 4.84 and 104.3 +/- 4.50 mmol/kg dry muscle in
horses receiving 0.25%,0.36%,0.45% and 0.56% dietary his,respectively. Dietary his did not (P>.05) affect intramuscular his or B-ala. In our study, plasma free-his increased with each dietary increment in his. Thus,plasma free his in the fed, mature horse was not a useful predictor of the his requirement. Furthermore,dietary his level did not influence intramuscular carn suggesting that a dietary level of 0.25% his is adequate to maintain intramuscular carn.
- ANDERSON, LH, ET AL. 1993. Investigations into the control of litter size in swine.II.Comparisons of morphological and functional embryonic diversity between Chinese and American breeds. J Anim Sci (Accept).
- MCGINNIS, LK & YOUNGS, CR. 1992. In vitro development of ovine embryos in CZB d medium. Theriogenology 37:559-569.
- PENDLETON, RJ, ET AL. 1992. Follicle stimulating hormone versus pregnant mare serum gonadotropin for superovulation of dairy goats. Small Rumin Res 8:217-224.
- PENDLETON, RJ, ET AL. 1992. Comparison of gluorogestone acetate sponges with norgestomet implants for induction of estrus and ovulation in anestrous dairy goats. Small Rum Res 8:269-273.
- YOUNGS, CR, ET AL. 1993. Investigations into the control of litter size in swine.I. Comparative studies on in vitro development of Meishan and Yorkshire preimplantation embryos. J Ani Sci (Accpt).
- YOUNGS, CR. 1992. Nuclear staining of sheep and pig embryos. In (P.J. Dziuk and M.B. Wheeler, Eds) Handbook of Methods for Study of Reproductive Physiology in Domestic Animals, pp V:B, 4.
- SMITH, RK, ET AL. 1992. Sequence determination of the displacement-loop region of bovine mitochondrial DNA from a single oocyte by using the polymerase chain reaction. J Dairy Sci 75:Sup 1:238.
- WEBER, PK, ET AL. 1992. An evaluation of potential vitrification solutions for.
Progress 01/01/91 to 12/30/91
In vitro culture of preimplantation embryos Early cleavage stage embryos from domestic farm animal species exhibit a block to further development when cultured in vitro. This developmental block is an artifact of in vitro culture systems and serves as a major obstacle in many embryo manipulation experiments. Previous studies indicated a detrimental effect of glucose on blastocyst formation in both sheep and pig embryos. A study was initiated to examine the effects of glutamine and lactate on in vitro development of sheep embryos. A total of 165 1- to 4-cell embryos was collected from 23 superovulated ewes. A modified CZB medium containing no glucose or EDTA served as the base medium and was modified to contain 0 or 1 mM glutamine and 0 or 31.3 mM sodium lactate in a 2x2 factorial treatment arrangement. Glutamine promoted blastocyst formation, whereas lactate had no effect. Lactate in the absence of glutamine, however, reduced blastocyst formation. Viability of embryos
cultured in lactate-free medium was demonstrated by establishment of pregnancy in 4/5 recipient ewes. An experiment was conducted to compare in vitro embryo development in prolific Chinese Meishan pigs with that of Yorkshire pigs. No difference in blastocyst formation rate was observed between breeds; however, Meishan embryos developed more slowly than did Yorkshire embryos. Meishan blastocysts contain fewer but larger cells than do Yorkshire blastocysts.
- MCGINNIS, L.K. and YOUNGS, C.R. 1992. In vitro development of ovine embryos in CZB medium. Theriogenology (in-press).
- PENDLETON, R.J., YOUNGS, C.R., RORIE, R.W., POOL, S.H., MEMON, M.A. and GODKE, R.A. 1992. Comparison of fluorogestone acetate sponges with norgestomet implants for induction of estrus and ovulation in anestrous dairy goats. Small Ruminant.
- PENDLETON, R.J., YOUNGS, C.R., RORIE, R.W., POOL, S.H., MEMON, M.A. and GODKE, R.A. 1992. Follicle stimulating hormone versus pregnant mare serum gonadotropin for superovulation of dairy goats. Small Ruminant Research (in-press).
- ANDERSON, L.H. and FORD, S.P. 1991. Investigations into the control of litter size: comparisons of Chinese and American Pigs. 1991 Swine Research Report, Iowa State University, A.S. Leaflet 857.
- CASEY, P.L., YOUNGS, C.R. and GODKE, R.A. 1990. Using computerized image analysis to evaluate cattle embryos before transfer. Louisiana Agriculture 33(4):9-12.
- MCGINNIS, L.K. and YOUNGS, C.R. 1991. In vitro culture of sheep embryos in CZB medium. 1991 Beef-Sheep Research Report, Iowa State University, A.S. Leaflet R842, pp 164-165.
- MCGINNISS, L.K. and YOUNGS, C.R. 1991. The use of human chorionic gonadotropin to enhance production of one-cell embryos from superovulated sheep. 1991 Beef-Sheep Research Report, Iowa State University, A.S. Leaflet R843, pp 165-16.
Progress 01/01/90 to 12/30/90
Culture of preimplantation embryos through the in vitro block to development: Early cleavage stage embryos from domestic farm animals exhibit a block to further development when cultured in vitro. This developmental block is an artifact of in vitro culture systems and serves as a major obstacle in many embryo manipulation experiments. A study was conducted to evaluate the ability of CZB medium to support the growth of 1- to 4-cell sheep embryos through the in vitro developmental block. A 2x2 factorial treatment arrangement was used to investigate the effects of glucose (5.56 mM vs 0 mM) and EDTA (0.11 mM vs 0 mM) on in vitro development. At the end of the 120 h culture period, embryos were stained with a DNA specific fluorochrome to enable counting of cell nuclei. EDTA had no effect on in vitro development, whereas glucose increased (P<.01) the incidence of fragmentation. Thirty-one percent of embryos cultured in glucose-free medium contained 30 or more nuclei, the
approximate number of cells in an in vivo developed blastocyst. Two experiments on the in vitro culture of 1- to 3-cell porcine embryos were conducted. In experiment 1, a 2x2 factorial treatment arrangement was used to examine the effects of glucose (5.56 mM vs 0 mM) and BSA (0.4% vs 1.5%) on in vitro development of embryos cultured in Whitten's medium. Embryos were cultured for 120 h and stained to enable counting of cell nuclei. The level of BSA had no effect on development; however, glucose inhibited (P<.01) blastocyst formation.
- MCGINNIS, LK. 1990. In vitro culture of ovine embryos in CZB medium. M. S. Thesis, Iowa State University, 71 pp.
- MCGINNIS, LK and CR YOUNGS. 1990. Vitrification of ovine embryos. Theriogenology 33:287.
- YOUNGS, CR and LK MCGINNIS. 1990. In vitro culture of porcine embryos in Whitten's medium containing varying levels of glucose and bovine serum albumin (BSA). Biology of Reproduction 42(Suppl 1):58.
- YOUNGS, CR, LK MCGINNIS, M JERNIGAN. 1990. Evaluation of progestagen and gonadotropin treatments for laparoscopic artificial insemination programs in sheep. 1990 Beef-Sheep Research Report, Iowa State University. A.S. Leaflet R744.
- YOUNGS, CR, LK MCGINNIS, SP FORD. 1990. In vitro culture of pig preimplantation embryos. 1990 Swine Research Report, Iowa State University, A.S. Leaflet R761, pp 34-35.
Progress 10/01/89 to 12/30/89
This is a new project iniitiated by a principal investigator who was recently hired. The principal investigator is only three months into the project, and the major effort to date has been procurement of laboratory equipment and other duties related to starting up a new lab. Preliminary experiments on the development of a defined culture system for cleavage stage ovine embryos have been initiated. Similar experiments aimed at culturing porcine embryos through the in vitro developmental block are presently underway.
- NO PUBLICATIONS REPORTED THIS PERIOD.
Progress 01/01/89 to 12/30/89
This is a new project initiated by a principal investigator who was recently hired. The principal investigator is only three months into the project, and the major effort to date has been procurement of laboratory equipment and other duties related to starting up a new lab. Preliminary experiments on the development of a defined culture system for cleavage stage ovine embryos have been initiated. Similar experiments aimed at culturing porcine embryos through the in vitro developmental block are presently underway.
- NO PUBLICATIONS REPORTED THIS PERIOD.