Effects of Differences in Dietary Protein and Varying the Interval from Collection of Bovine Embryos to Freezing on Embryo Quality and Viability

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Virginia Tech


High levels of dietary protein may be detrimental to reproductive performance in cattle. The objective of Exp. 1 was to determine the effects of differences in dietary protein on the production and quality of bovine embryos collected from superovulated donors. Angus cows were randomly assigned to receive one of three experimental diets: a daily ration of 5.7 kg poultry litter, 2.0 kg hay, 3.1 kg corn, and 0.5 kg peanut hulls (LITTER; n = 15); a daily ration of 6.2 kg peanut hulls, 2.2 kg soybean meal, 2.0 kg hay, 0.5 kg corn, and 0.4 kg dicalcium phosphate (SBM; n = 15); or a daily ration of 6.2 kg peanut hulls, 2.0 kg hay, and 3.1 kg corn (CON; n = 19). Diets differed in the amount of total, soluble and degradable protein, but were comparable in energy. After 30 d on the diets, all cows were treated to induce superovulation (28.8 mg FSH/cow, Folltropin) and synchronize estrus. After the detection of estrus each cow was inseminated with semen from one of four Holstein bulls. Embryos were collected 7 d after estrus and evaluated for quality (according to the International Embryo Transfer Society (IETS) standards) and stage of development. Prior to treatment to induce superovulation, blood samples were collected 6 h after feeding. Samples were analyzed to assess dietary effects on plasma urea nitrogen (PUN). Mean levels of PUN were higher (P < 0.01) in cows fed the LITTER or SBM diet (16.3 mg/dL, LITTER; 21.8 mg/dL, SBM; 9.7 mg/dL, CON) than in cows fed the CON diet. Additionally, concentration of PUN was higher in cows fed SBM than in those fed LITTER (P < 0.01). An average of 9.2 transferable embryos (Grade 1, 2 and 3) was collected from each cow and there were no significant differences in the number of transferable embryos collected among groups (9.2, LITTER; 9.3, SBM; 9.1, CON). The number of degenerate embryos or unfertilized ova did not differ among dietary groups. High-protein diets elevated PUN, but did not affect the number or quality of embryos collected from superovulated donors.

Cryopreservation of bovine embryos is an important aspect of a successful embryo transfer program. The objective of Exp. 2 was to evaluate the post-thaw viability of bovine embryos collected in Exp. 1 in an in vitro culture system after the embryos had been held at room temperature or refrigerated for 2 to 12 h prior to freezing. Upon embryo recovery, each embryo was randomly assigned to be placed in holding media for 2, 6 or 12 h prior to freezing. During this interval, one-half of the embryos were maintained in a refrigerated environment (5 °C), while the remaining half of the embryos were held at room temperature (20.5 to 22 °C) until freezing. Immediately prior to freezing, embryos were removed from the holding media, transferred to a well containing ethylene glycol (10%) in ovum culture media and loaded individually into a 0.25-mL plastic straw. Straws were then placed in a freezer unit (-6 °C) and seeded to induce ice crystal formation through all columns of the straw. The temperature of the freezer was then decreased 0.6 °C/min to -32 °C, and straws were loaded into canes and plunged into a liquid nitrogen tank (-196 °C). After storage, each straw was exposed to a 5-s air thaw and placed in a water bath at 35 °C for 20 s. Each embryo was then washed to remove excess ethylene glycol prior to in vitro culture. Embryos were individually cultured in Ham's F-10 media supplemented with 4% fetal bovine serum for 72 h. Embryos were evaluated at 24 h intervals throughout the culture period and assigned a stage of development and quality grade score (according to IETS standards). The percentage of embryos that developed to the expanded blastocyst stage and hatched from the zona pellucida was greater for embryos held 2 or 6 h prior to freezing (P < 0.05) than for embryos held for 12 h after collection before being frozen (62.9, 52.0 and 31.1%, respectively). The percentage of embryos that degenerated during in vitro culture was lower for embryos held 2 or 6 h prior to freezing (20.4 and 26.6%; P < 0.05) than for embryos held for 12 h before freezing (50.8%). Furthermore, embryo quality grade was more desirable for embryos held for 2 or 6 h (1.5 and 1.7; P < 0.05) than for those held for 12 h before freezing (2.1). The semen used to inseminate donors and the diet fed to donors for 4 wk prior to embryo collection did not influence the proportion of embryos that hatched or degenerated during the 72 h of in vitro culture. Additionally, holding embryos in a refrigerated environment from the time of collection until freezing did not enhance embryonic development during post-thaw culture. Thus, embryonic viability may be impaired when embryos are held longer than 6 h following embryo recovery before being frozen; however, the storage temperature during the interval from collection to freezing does not influence embryonic development post-thaw.



Superovulation, Bovine, Embryo, Dietary Protein, Cryopreservation