GERM CELL AND EMBRYO DEVELOPMENT AND MANIPULATION FOR THE IMPROVEMENT OF LIVESTOCK

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3399	1050	50%
301	3599	1050	50%

Progress 10/01/99 to 09/30/04

Outputs
Studies assessed the effectiveness of various treatments to synchronize nuclear and cytoplasmic maturation of porcine oocytes. Results indicated that dexamethasone and dibutyryl cAMP can be used to delay nuclear maturation without detrimental effects on subsequent maturation and cleavage. The time of gonadotropin addition during maturation influenced subsequent maturation and cleavage of porcine oocytes. Experiments investigating the effects of vitamins A and E on boar sperm motility, acrosome integrity, viability, and oxidation found that vitamin C and E supplementation decreased oxidation of sperm membranes, increased sperm motility and post-thaw viability. A study examined the toxic effects and possible mechanisms of fescue ergot alkaloid toxins on bovine sperm function and motility. Results indicated that ergot toxins can inhibit sperm motility in a concentration and time dependent manner. Possible mechanisms for toxicity were altered intracellular pH and protein synthesis. A study evaluating the use of sexed bovine semen for in vitro embryo production found that cleavage and subsequent embryo development were similar for X- or Y-bearing spermatozoa but development rate and morphological quality were reduced when compared to unsexed semen... A study evaluating the effect of sire and breed on sex ratio of calves born found no overall differences among breeds in the ratio of male to female offspring. Within breeds, some sires consistently produced more male or female offspring over time. Cows conceiving early in the breeding season produced more heifer calves whereas cows conceiving late in the season produced more bulls. Another study evaluating the effects of estrus parameters on AI pregnancy rates found that the interval from synchronization treatment to estrus, length of estrus and number of mounts during estrus were similar among cows that conceived and failed to conceive. A study evaluating the effect of embryo-recipient synchrony on transfer pregnancy rate found that selection of recipients with synchrony of plus or minus 12 hours can improve embryo transfer pregnancy rates. Other studies evaluating estrous synchronization schemes for embryo recipients found that estradiol can be substituted for GnRH in this estrous synchronization scheme without detrimental effects on subsequent corpus luteum function or pregnancy rate. A study comparing dinoprost with cloprostenol for inducing estrus in cattle found both products to be equal in their effectiveness in inducing estrus in beef cows and heifers. The interval from treatment to estrus and intensity of estrus were also similar for both products. A behavioral study found that social hierarchy of dairy heifers influences the length and expression of estrus but not fertility. Other studies have evaluated the innate resistance of individual cows or bulls to pesticide-resistant flies and could lead to identification of genes as candidates for the production of transgenic animals with resistance to external parasites. Current studies will continue for this revised project (W-1171) under Arkansas project, ARK02054.

Impacts
Studies to improve the efficiency of bovine oocyte maturation, fertilization and embryo development procedures in conjunction with sexed semen could lead to increased adoption of this technology by producers. Identification of other factors contributing to the sex ratio of offspring may lead to procedures for producers to increase the number of offspring of the desired sex. Biotechnologies such as transgenic animal production and cloning are dependent on in vitro embryo production procedures. Improvement in the efficiency of these underlying embryo production procedures will improve the efficiency and application of related technologies for the improvement of livestock. Studies show that antioxidants may improve function and post-thaw viability of spermatozoa and could expand the availability of superior genetics to producers. Results of embryo transfer studies could lead to improvement in transfer pregnancy rates and increase efficiency of procedures for synchronization of estrus between embryo donors and recipients. Investigations into the effects of estrus parameters on fertility should lead to insight into factors influencing artificial insemination and embryo transfer pregnancy rates. Endophyte-infected fescue is known to contain ergot alkaloids that adversely effect female reproduction, but little is known of their possible effects on male reproduction. Current studies provide insight into the effects of ergot alkaloids on sperm function and should lead to a better understanding of overall sperm physiology.

Publications

Nihsen, M.E., Piper, E.L., West, C.P., Crawford, R.J., Denard, T.M., Johnson, Z.B., Roberts, C.A., Spiers, D.A., and Rosenkrans, C.F. 2004. Growth rate and physiology of steers grazing tall fescue inoculated with novel endophytes. J Anim Sci. 82:878-83.
Kreider, D., Post, N., Rorie, R., Lester, T. and. French, E. 2004. MGA and CIDR based timed AI protocols in postpartum beef cows. J. Anim. Sci. 82(Suppl. 1):255.

Progress 01/01/03 to 12/31/03

Outputs
A study evaluated the effect of sire and breed on sex ratio of calves born over a period of 15 to 33 years in five breeds of beef cattle. There were no overall differences among breeds in the ratio of male to female offspring. Within breeds, some sires consistently produced more male or female offspring over time. Cows conceiving early in the breeding season produced more heifer calves whereas cows conceiving late in the season produced more bulls. Studies have evaluated the innate resistance of individual cows or bulls to pesticide-resistant flies. These studies could lead to identification of genes as candidates for the production of transgenic animals with resistance to external parasites. Studies are underway to assess the effects of seminal plasma proteins on the viability of boar and bull spermatozoa during and after cryopreservation. Also, alternative cryopreservation procedures, such as vitrification will be evaluated. Under the revised project, a series of studies will be initiated to investigate the use of microchannel devices (VitaeCell) for all aspects of embryo production in vitro. Experiments will continue to evaluate the effects of ascorbic acid and alpha-tocopherol on pre- and post-thaw survival and morphology of boar spermatozoa.

Impacts
Identification of factors contributing to the sex ratio of offspring could lead to procedures for producers to increase number of offspring of the desired sex. Studies leading to improvement in the post-thaw viability of bull and boar spermatozoa could expand the availability of superior genetics to producers. Adoption of microfluidic technology to in vitro embryo production has the potential to simplify in vitro procedures while improving efficiency and repeatability.

Publications

Baublits, R.T., Brown, Jr., A.H., Johnson, Z.B., Rosenkrans, Jr., C.F., Rorie, R.W. and Sandelin, B.A. 2003. Variation in the percentage of female births over time in five breeds of registered cattle. Prof. Anim. Sci. 19:204-210.
Steelman, C.D., et al. C.F. 2003. Evaluation of alternative tactics for management of insecticide-resistant horn flies (diptera muscidae). J. Econ. Entomol. 96:892-901.

Progress 01/01/02 to 12/31/02

Outputs
Experiments were conducted to investigate the effects of Vitamins A and E on boar sperm motility, acrosome integrity, viability, and oxidation when incubated at 39 C for 24 hours (Exp 1), stored at 18 C for 3 days (Exp 2) or cryopreserved (Exp 3). The main effects were Vit C (0, 0.1, 1.0, 10.0 mM) and Vit E (0, 0.1, 1.0, 10 mM). For Experiment 1, significant interactions between main effects and hour of incubation occurred. At the end of the 24 hour incubation, control sperm had lower motility than sperm incubated in 0 mM Vit C and 10 mM Vit E (7.4 vs. 31.8 percent). Oxidation of sperm, as represented by TBARs assay, demonstrated a bimodal response during the 24 hour incubation. Overall, inclusion of Vit C and E decreased oxidation of sperm membranes. Day of storage was a significant source of variation for Experiment 2. Inclusion of 10 mM Vit C and 0.1 mM Vit E increased sperm motility on day 3 when compared with controls (72.6 vs. 14.2 percent). Together or alone, Vit C and E decreased sperm membrane oxidation during all days of storage. Results from Experiment 3 demonstrated post-thaw motility was increased by 10 mM Vit C in combination with 0.1, 1.0, or 10.0 mM Vit E. Percentage of intact acrosomes increased when 10.0 mM Vit C and 0.1,1.0, and 10.0 mM Vit E were in the freezing medium. Viability, as evaluated by HOST assay, increased with the addition of Vit C and Vit E while spermatozoa oxidation, as measured by TBARs, was decreased. These results demonstrate that Vit C and E enhance physiological characteristics of boar spermatozoa. Another study with cattle compared the effectiveness of two prostaglandin F2alpha products, Lutalyse (dinoprost) and Estrumate (cloprostenol), for induction of estrus in beef cattle. A total of 385 Angus or crossbred beef cows and heifers from three herds were used in the study. Within herds, the cows or heifers were randomly selected to receive the recommended dosage of either dinoprost or cloprostenol to induce estrus. All animals were continuously monitored for estrus, using a HeatWatch electronic mount monitoring system. The time of treatment was recorded so that the interval from treatment to onset of estrus could be determined. The results of this study indicate that dinoprost or cloprostenol are equal in their effectiveness in inducing estrus in beef cows and heifers. A similar percentage (67 to 70 percent) of animals were detected in estrus after treatment, regardless of the estrous synchronization scheme in which the products were used. The interval from treatment to estrus and intensity of estrus were similar for both products. With both dinoprost and cloprostenol, the number of animals in estrus peaked about 60 hours after treatment.

Impacts
The studies with the antioxidants, Vitamin C and E should lead to better understanding of the causes of impaired function of boar sperm occurring during storage, leading to treatments to prevent altered sperm function and thus, improve the fertility of swine artificially inseminated with stored or cryopreserved semen. The study with dinoprost and cloprostenol provides producers with unbiased information on the effectiveness of these products for synchronization of estrus in beef cows and heifers.

Publications

Burke, J.M. and Rorie, R.W. 2002. Changes in ovarian function in mature beef cows grazing endophyte-infected tall fescue. Theriogenology 57:1733-1742.
Golden, C D. 2002. Effects of ascorbic acid and alpha-tocopherol on physiological characteristics of boar spermatozoa. MS Thesis, University of Arkasas, Fayetteville.
Golden, C, Rosenkrans, C., Jr., and Johnson, Z. 2002. Effects of ascorbic acid and alpha-tocopherol on cryopreserved boar sperm. Arkansas Animal Science, Research Series 499:120-123.
Looper, M.L., Rosenkrans, C.F., Jr., Johnson, Z.B., Brown, A.H., Jr., Hornsby, J.A. and Perkins, J.L. 2002. Relationships among growth characteristics of replacement Angus heifers. The Prof. Anim. Scientist 18:120-126.
Melnychuk, V.L., Cooper, M.A., Kirby, J.D., Rorie, R.W. and Anthony, N.B. 2002. Use of ultrasonography to characterize ovary status in chickens. J. Poultry Sci. 81:892-895.
Post, N.M., Kreider, D.L., Rorie, R., Lester, T. and Cole, K. 2002. Estrus synchronization and timed artificial insemination with melengesterol acetate plus GnRH in beef females. Arkansas Animal Science, Research Series 499:126-129
Rorie, R.W., Bilby, T.R. and Lester, T.D. 2002. Application of electronic estrus detection technology to reproductive management of cattle. Theriogenology 57:137-148.
R.W. Rorie, Bilby, T.R. and Lester, T.D. 2002. Comparison of Electronic Mount Monitors for Detection of Estrus in Cattle. Arkansas Animal Science, Research Series 499:124-125.
R.W. Rorie and Lester, T.D. 2002. Comparison of Lutalyse and Estrumate for Inducing Estrus in Beef Cows and Heifers. Arkansas Animal Science, Research Series 499:130-131.

Progress 01/01/01 to 12/31/01

Outputs
The most critical factor determining the success of an artificial insemination (AI) program in cattle is estrus detection. Studies show that less than half of dairy cows are detected in estrus by visual observation. A study was conducted to directly compare the effectiveness of electronic methods (HeatWatch, MountCount and ShowHeat) for estrus detection in dairy heifers. The MountCount and ShowHeat monitors were found to require more labor and animal handling than the HeatWatch system. However, all three systems were equally effective in detecting estrus. Over a 28-day period, all of the heifers were detected in estrus by the electronic mount monitors. Heifers detected in estrus were artificially inseminated, resulting in a first service pregnancy rate of 70 to 72 percent and an over pregnancy rate of 93 to 96 percent.

Impacts
This study illustrates the effectiveness of electronic mount monitors for estrus detection in cattle. Adoption of electronic estrus monitoring technology by producers has the potential of increasing the use and reducing the overall costs of artificial insemination, by improving estrus detection efficiency and reducing the number of services per conception.

Publications

Brown, A. H., Kellogg, D. W., Johnson, Z. B., Rorie, R. W. Coblentz, W. K. Sandelin, B. A. Lesmeister K. E. and Jackson, W. R. 2001. Growth, luteal activity and pregnancy rates of three breed types of dairy heifers in a forage-based development program. Prof. Anim. Sci. 17:20-26

Progress 01/01/00 to 12/31/00

Outputs
A study assessed the effectiveness of various treatments to prevent porcine oocyte germinal vesicle (GV) breakdown for a period of 20 h without detrimental effects to subsequent maturation and cleavage. Maturation medium with FSH was used alone (control), or supplemented with either dexamethasone (DEX), dimethylaminopurine (DMAP) or dibutyryl cyclic adenosine monophosphate plus testosterone (dbcAMP plus T). After the initial 20 h of culture, oocytes were transferred to medium with LH for an additional 26 h of maturation. Results indicated that DMAP irreversibly blocked GVBD, with few oocytes maturing to MII or cleaving after activation. Both DEX and dbcAMP plus T treatments were reversible and resulted in similar rates of maturation to MII and cleavage. When compared to the control group, all treatments reduced cleavage. Another study investigated whether the presence or absence of gonadotropins during the first or second half of in vitro maturation affected porcine oocyte maturation and cleavage after activation. Oocytes were matured in medium without hormones, with FSH and LH the first 20 h only, FSH for 20 h and LH for 26 h or FSH and LH the last 26 h of maturation. The results indicated that timing of gonadotropin supplementation can influence maturation and subsequent cleavage of porcine oocytes. Delaying FSH and LH supplementation until after 20 h of maturation reduced maturation of oocytes to MII and cleavage. A study was conducted to evaluate the effect of embryo-recipient synchrony on transfer pregnancy rate. All embryo donors and recipients were monitored with a HeatWatch system to determine exact estrous synchrony. Recipient synchrony was categorized in 12 h intervals to evaluate the effect of donor-recipient synchrony on pregnancy rate. Pregnancy rates were similar when compared at 12 h intervals from minus to plus 24 h. However, the pregnancy rate was higher for synchrony of plus/minus 0 to 12 h than that of plus/minus 12 to 24 h (60 versus 46 percent, respectively). The results of this study suggest that continuous monitoring of embryo donors and recipients and selection of recipients with synchrony of plus/minus 12 h can improve embryo transfer pregnancy rates.

Impacts
Improving the synchrony between nuclear and cytoplasmic maturation might improve oocyte developmental competence. We have also found that the timing of gonadotropin supplementation can influence maturation and subsequent cleavage of porcine oocytes. Our studies should lead to new procedures to improve the developmental competence of porcine oocytes and embryos. Other results show that embryo transfer pregnancy rates in cattle can be improved by electronically monitoring for estrus in embryo donors and recipients and transferring embryos to recipients with asynchrony of no more than plus/minus 12 h.

Publications

Bilby, T R and Rorie, R W 2001. Effectiveness of maturational treatments to block porcine oocyte germinal vesicle breakdown without reducing subsequent nuclear maturation and cleavage. Theriogenology 55:463 (Abstr)
Bilby, T R and Rorie, R W 2000. Effects of sequence and timing of gonadotropin supplementation on porcine oocyte maturation and subsequent cleavage in vitro. Southern Section, American Society of Animal Science (accepted).
Melnychuk, V.L., Cooper, M.A., Rorie, R.W., Kirby, J.D. and Anthony, N.B. 2000. Characterization of ovary status using ultrasound techniques. J. Poultry Sci. (accepted)
Kreider, D L, Rorie, R, Brown, D, Miller, F and Wright, S 2000. Enhancement of ovulation rate and litter size in gilts. J. Anim. Sci. (accepted)
Brown, A H, Kellogg, D W, Johnson, Z B, Rorie, R W, Coblentz, W K, Sandelin, B A, Lesmeister, K E and Jackson, W R. 2000. Growth, luteal activity and pregnancy rates of three breed types of dairy heifers in a forage based development program. Prof. Anim. Sci. (accepted)
Brown, A H, Kellogg, D W, Johnson, Z B, Rorie, R W, Coblentz, W K, Lesmeister, K E and Jackson, W R. 2000. Growth, luteal activity and pregnancy rates of three breed types of dairy heifers. J. Anim. Sci. 78(Suppl 1):54 (Abstr)

Progress 01/01/99 to 12/31/99

Outputs
Studies have been initiated to determine if delaying germinal vesicle (GVBD) breakdown by exposure of bovine oocytes to dibutyryl cyclic AMP during in vitro maturation will increase the percentage of female embryos incorporating x-bearing spermatozoa at fertilization, without a reduction in oocyte developmental competence. The treatments include fertilization after 16 hours of maturation, fertilization after 24 hours of maturation, and oocytes cultured with dibutyryl cyclic AMP for the first 8 hours and fertilized at 24 hours. At this time, early cleavage stage embryos derived from each treatment have been frozen and will be sexed in the near future. Another study has been initiated to determine if delaying GVBD in porcine oocytes will reduce the incidence of polyspermy and increase developmental competence. Porcine oocytes were cultured in M-199 supplemented with and without dibutyryl cyclic AMP and testosterone. After the initial 20-hour culture period, the oocytes were cultured an additional 24 hours in M-199 containing serum and hCG. Dibutyryl cyclic AMP and testosterone treatment appeared to be effective in preventing GVBD, with about 80% of the oocytes still at the GV stage at 20 h. Both treatments resulted in about 75% of the oocytes achieving MII by 44 hours of IVM. Further studies will evaluate the development of porcine oocytes after fertilization and/or parthenogenic activation. A study evaluated the effect of interval from synchronization treatment to onset of estrus, intensity (mounting activity) and length of estrus, and timing of insemination on pregnancy rate in beef cattle. The interval from synchronization treatment to onset of estrus ranged from 4 to 147 hours. The interval from synchronization treatment to onset of estrus had no affect on subsequent pregnancy rate. Cows detected in estrus were artificially inseminated between 7.5 and 26 h after the onset of estrus. Pregnancy rates were similar among insemination times. These results suggest that when the actual onset of estrus is known, timing of inseminations can be adjusted for convenience (within reason) without compromising pregnancy rate. The period of standing estrus of individual cows and heifers ranged from less than 1 hours to almost 24 hours in this study. Pregnancy rate was similar regardless of length of estrus. Mounting activity ranged from 4 to over 147 mounts during estrus. Numerically, pregnancy rate was higher for animals with 30 to 50 total mounts during estrus. However, there was no statistical difference in pregnancy rate due to mounting activity. Comparison of all pregnant versus open cows, reveal a trend for cows with fewer mounts during estrus to have reduced fertility. The interval from synchronization treatment to estrus, length of estrus and interval from onset of estrus to insemination were similar for open and pregnant cows.

Impacts
In vitro embryo production procedures remain inefficient and typically result in a higher percentage of male offspring. Our research should lead to procedures for producing a higher percentage of female embryos while improving oocyte developmental competence. Our investigations into the effects of estrus parameters on fertility should lead to insight into factors influencing artificial insemination and embryo transfer pregnancy rates.

Publications

Rorie, RW, TD Lester, BR Lindsey and RW McNew. 1999. Effect of timing of artificial insemination on gender ratio in cattle. Theriogenology 52(6):1035-1041.
Rorie, RW. Effect of timing of insemination on mammalian sex ratio. 1999. Theriogenology 52(8)1273-1280.
Lester, TD, RW McNew and RW Rorie. 1999. Use of electronic estrous detection to evaluate the effect of embryo-recipient synchrony on pregnancy rate in cattle. Theriogenology 51(1):265 (Abstr).
Lorton, LL, T. Lester, CF Rosenkrans, Jr. and RW Rorie. 1999. Effect of estrous parameters and time of insemination on pregnancy rate in beef cattle. . J. Anim. Sci. 77(Suppl 1):15 (Abstr 59).
Rorie, RW, TD Lester, BR Lindsey and RW McNew. 1999. Effect of timing of artificial insemination on gender ratio in cattle. Theriogenology 51(1):299 (Abstr).

Progress 01/01/98 to 12/31/98

Outputs
A study investigated whether time of insemination can be used to alter sex ratio in cattle. Beef heifers and cows (n = 98) were synchronized, monitored electronically for estrus and inseminated at either 10 or 20 hours after estrus. Sixty to 80 days after insemination, ultrasonography was used to confirm pregnancy status and to determine fetal sex. Time of insemination had no effect on pregnancy rate. The overall pregnancy rate was 69 percent. Time of insemination had no effect on sex ratio of offspring, with 54 and 52 percent of the calves born from the early and late inseminations being bulls, respectively. A study evaluated the effect of embryo donor and recipient synchrony on transfer pregnancy rate. Embryo donors (n = 20) and recipients (n = 146) were electronically monitored for estrus so that exact synchrony could be determined. Embryos were transferred to recipients within 24 hours synchrony of donors. Pregnancy was confirmed at 45 to 60 days of gestation. Pregnancy rate was higher for donor-recipient synchrony of 12 than 24 hours (58 versus 48 percent, respectively. Results indicate embryo transfer pregnancy rate can be improved by closer synchrony between the embryo donor and recipient than is routinely used. A study evaluated the effect of interval from synchronization treatment to onset of estrus, length and intensity (mounting activity) of estrus, and timing of insemination on pregnancy rate in beef cattle. An electronic estrous detection system was used to continuously monitor for onset of estrus in beef cows. Cows detected in estrus (n = 176) were artificially inseminated between 7.5 and 25.3 hours after the onset of estrus. Overall pregnancy rate was 66.5%. The interval from synchronization treatment to onset of estrus ranged from 4 to 147 hours. Pregnancy rate was not effected by the interval from synchronization treatment to estrus. Length of estrus ranged from .5 to 23.3 hours. Mounting activity ranged from 3 to 154 mounts per estrus period. Neither length nor intensity of estrus affected pregnancy. Pregnancy rate was not affected by timing of insemination. Knowing the actual onset of estrus allows for flexibility in time of insemination without compromising pregnancy rate.

Impacts
(N/A)

Publications

Lester, T.D., McNew, R.W. and Rorie, R.W. 1999. Use of electronic estrus detection to evaluate the effect of embryo-recipient synchrony on pregnancy rate in cattle. Theriogenology 51:265.
Lorton, L.L., Lester, T.D., Rosenkrans, Jr. C.F. and Rorie, R.W. 1999. Effect of estrous parameters and time of insemination on pregnancy rate in beef cattle. Proc., Am. Soc. Anim. Sci., So. Sec., p 15, abstr 59.
Rorie, R.W., Lester, T.D., Lindsey, B.R. and McNew, R.W. 1999. Effect of timing of insemination on sex ratio in beef cattle. Theriogenology 51:399.

Progress 01/01/97 to 12/31/97

Outputs
Studies are underway to evaluate the effects of serum supplementation on secretion of oviductal and uterine proteins in vitro. Oviductal and uterine epithelial cultures will be established and exposed to serum collected from open and pregnant cows on day 0, 4, 8 and 16 of the estrous cycle. Protein secretion patterns will be compared to determine if secretion can be altered in vitro by serum supplementation. Any stage specific proteins will be isolated and added to defined culture medium to determine their effects on embryo development. Studies will also be initiated on porcine oocyte maturation, fertilization and embryo culture in vitro.

Impacts
(N/A)

Publications

No publications reported this period

Progress 01/01/96 to 12/30/96

Outputs
The effect of 5, 10 or 20% atmospheric oxygen on bovine oocyte maturation, fertilization and embryo culture in an oviductal co culture system was evaluated. The cleavage rate of oocytes 20% oxygen was higher than after IVM under reduced oxygen of either 5 or 10% oxygen. Development to the blastocyst stage was higher for embryos matured under reduced oxygen (5%). Cleavage rates and development rates were similar when bovine embryos were fertilized in either a 5, 10, or 20% oxygen atmosphere. Development to the blastocyst stage was significantly greater when embryos were cultured under reduced (5%) oxygen when compared to either 10 or 20% oxygen. The effect of serum supplementation on secretion of oviductal cells used for embryo culture was evaluated. Embryos were cultured with oviductal cells in phenol red free M 199 supplemented with 20% 1) FCS for 0 to 144 h, 2) D0 serum for 0 to 144 h, 3) D0 serum for 0 to 48 h, and D 4 serum from 48 h to 144 h, 4) D0 serum for 0 h to 48 h, and D 8 serum from 48 h to 144 h, or 5) D0 serum for 0 to 48 h, then D 4 from 48 h to 96 h, and D 8 from 96 h to 144 h. A higher percentage of 3 to 4 cell embryos developed to the blastocyst stage in treatments 4 and 5 compared to treatment 1 or 2. The number of cells per blastocyst was highest in media containing Day 8 or a series of Day 0 Day 4 Day 8.

Impacts
(N/A)

Publications

MILLER, G.F. 1996.
EFFECTS OF OXYGEN CONCENTRATIONS IN THE GAS ATMOSPHERE ON BOVINE OOCYTE MATURATION, FERTILIZATION AND EMBRYO DEVELOPMENT IN VITRO. PH.D. THESIS. NASTI, K.B. 1996. BOVINE OVIDUCTAL EPITHELIAL CELL PROTEIN SECRETION, AND ITS EFFECT ON EMBRYO DEVELOPMENT IN VITRO. PH.D. THESIS.

Progress 01/01/95 to 12/30/95

Outputs
A study determined if protein secretion by oviductal cells could be altered by medium supplementation with serum collected from cattle at different stages of the estrous cycle. Oviductal cells were cultured in medium with serum from blood collected on day 0. 2, 4, 6 and 8 post estrus. Secretion of 10 of 20 proteins detected were altered by serum supplementation. similar studies with uterine cells indicated that protein secretion patterns were unaltered, regardless of serum supplementation. In another study, bovine oocytes were matured under atmospheres of 5, 10 or 20% oxygen. The development rate of cleaved embryos to the blastocyst stage indicated that reducing the oxygen atmosphere to 5% during bovine oocyte maturation may enhance oocyte developmental competence. A similar study compared the effect of varying oxygen concentration during in vitro fertilization on subsequent developmental competence of in vitro matured bovine oocytes. Cleavage tended to be reduced when oocytes were fertilized in the presence of reduced oxygen, but the percentage of cleaved embryos developing to the blastocyst stage and the number of cells per blastocyst were not affected by treatment.

Impacts
(N/A)

Publications

Progress 01/01/94 to 12/30/94

Outputs
This project was revised in the past year and new studies have been initiated. Astudy is underway to investigate the effects of 5, 10 or 20% oxygen atmosphere on maturation, fertilization and cleavage rate of bovine oocytes in vitro. The first study, comparing oocyte maturation rates in different atmospheres, is complete and data is being analyzed. Another study is underway to determine if protein secretion by oviductal cells can be altered by medium supplementation with serum collected from cattle at different stages of the estrous cycle. Results indicate that secretion of at least 10 proteins was altered by supplementing medium with the different sera. The affects of this altered protein secretion pattern on in vitro embryonic development will be investigated.

Impacts
(N/A)

Publications

Progress 01/01/93 to 12/30/93

Outputs
A study was conducted to determine if citrate was embryotrophic to bovine embryos. Results indicated that the variation noted for the ability of different BSA lots to support embryo development is not correlated with citrate concentration. Also, addition of 0, 100, 300 or 900 micromolar of citric acid to define medium did not improve embryo development to the blastocyst stage. Other studies indicate embryo development to the blastocyst stage is superior when embryos are cultured with oviductal cells in serum supplemented synthetic oviductal fluid medium rather than Medium 199. A study have been initiated to determine the affects of antioxidants on bovine oocyte maturation, fertilization and embryo culture. Different types of somatic cells used for embryo culture are being compared to determine if there are similarities in protein secretions. Studies have been initiated to identify factors contributing to oocyte sensitivity to cooling.

Impacts
(N/A)

Publications

RORIE R.W., MILLER, G.F., NASTI, K.B. 1994. Effects of citrate on development of bovine embryos in defined medium. J. Anim. Sci. ACCEPT OF PUB.
GODKE, R.A., RORIE, R.W. 1993. Embryo microsurgery for large animals. In: Gamete and Embryo Micromanipulation in Human Reproduction. S. Fishel and E. Symonds (Eds.) Edward Arnold, Kent, England. Chapter 15, pp. 262-272.
LESTER, T.D. 1993. Post thaw viability of bovine trophoblastic vesicles cryopreserved in either glycerol, dimethyl sulfoxide, propylene glycol or ethylene glycol. M.S. THESIS.
LESTER, T.D., MILLER, G.F., ET AL. 1993. Post thaw viability of bovine trophoblastic vesicles cryopreserved in either glycerol, dimethyl sulfoxide, propylene glycol or ethylene glycol. Theriogenology ACCEPT FOR PUB.
MILLER, G.F., ET AL. 1993. Addition of (BOEC) and/or penicillamine, hypotaurine and epinephrine (PHE) to bovine in vitro fertilization (IVF) medium increases the subsequent embryo cleavage rate. Ther. IN PRESS.
MILLER, G.F., MCNEW, R.W., RORIE, R.W. 1994. Effects of hyaluronidase on cleavage of bovine oocytes. J. Dairy Sci. ACCEPT FOR PUB.

Progress 01/01/92 to 12/30/92

Outputs
Addition of bovine oviductal epithelial cells (BOEC) and/or PHE to fertilizationmedium of in vitro matured bovine oocytes with addition of PHE and/or BOEC to IVF medium increased subsequent embryo cleavage rate. Embryos fertilized with either/both were more advanced at 45 hours post insemination. Evaluation on development of in vitro produced bovine embryos in defined medium (Seidel's B2- 2) supplemented with 0, 100, 300 (for entire culture period or added on day 3 of culture) or 900 #M of citric acid. Numbers of embryos developed to the blastocyst stage in medium containing citrate were similar, regardless of level of citrate supplementation. Use of glycerol, dimethyl sulfoxide, propylene glycol and ethylene glycol as cryoprotectants for bovine trophoblastic vesicles (BOEC and PHE) was studied. Ethylene glycol was less toxic and afforded better cryoprotection to bTV than other cryoprotectants evaluated. Ovulation rate in FSH-P stimulated donor cows immunized against dehydroepiandrosterone (DHEA) conjugated to human serum albumin. Increases in mean number of recovered and viable embryos in cows immunized against DHEA and ovulation rate in primiparous gilts was determined after immunization against: Control; injection vehicle only; androstenedione; inhibin a subunit; androstenedione plus inhibin a subunit; 17a hydroxyprogesterone; and dihydroepiandrosterone. All immunization treatments except inhibin increased ovulation rate over the control group.

Impacts
(N/A)

Publications

Progress 01/01/91 to 12/30/91

Outputs
A series of experiments was conducted to optimize oocyte maturation, fertilization, storage and culture methods. We have shown enzymatic removal of cumulus cells prior to fertilization, with hyaluronidase, reduces the subsequent cleavage rate. This reduction in cleavage rate is eliminated by the addition of trypsin inhibitor to the hyaluronidase preparation. Preliminary results of a culture study indicate there are no beneficial effects to the sequential supplementation of embryo culture medium with serum collected from cows on days of the estrous cycle corresponding to the embryo development stage in vitro. Another study confirmed that BSA (bovine serum albumin) lot variation can significantly influence bovine embryo development in simple culture medium. Available BSA lots should be screened before their inclusion in culture medium for bovine embryos. However, preliminary data indicates that BSA lot variation is not a major factor influencing the development of early-stage porcine embryos in simple medium. Bovine embryo development to the morula or blastocyst stage was compared in a study where synthetic oviductal fluid was supplemented with either BSA or FCS (fetal calf serum).

Impacts
(N/A)

Publications

RORIE, R.W., ET AL. 1991. In vitro development of bovine embryos cocultured with oviductal epithelial cells in either synthetic oviductal fluid or M-199 medium. Theriogenology (in-press).
GLIEDT, D.W., ET AL. 1991. Development of in vitro produced bovine embryos co-cultured with oviductal cells in M-199 medium. J. Anim. Sci. 69 (Suppl. 1):48.
MILLER, G.F., ET AL. 1991. Addition of trypsin inhibitor to hyaluronidase preparations used for oocyte cumulus cell removal prior to in vitr fertilization. J.Anim. Sci. (Suppl. 1):47
RORIE, R.W., ET AL. 1991. Development of in vitro produced bovine embryos in synthetic oviductal fluid medium with different lots of fraction V bovine serum albumin. J. Anim. Sci. 69 (Suppl. 1):47.
RORIE, R.W., ET AL. 1991. In vitro development of bovine embryos cultured in synthetic oviductal fluid medium suppl. with fetal calf serum or bovine serum albumin. J. Anim. Sci. 69 (Suppl. 1):402.