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Repeat-breeding is one of the important factors that affect dairy management. The objective of this study was to investigate the effect of transfer of frozen–thawed IVF embryos on pregnancy in repeat-breeder Holstein cattle. Cumulus–oocyte complexes (COCs) were collected by aspiration of 2–1-mm follicles from ovaries obtained at a local abattoir. COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg/mL of FSH at 38.5°C under a 5% CO2 atmosphere in air. Matured oocytes were inseminated with spermatozoa of 5 × 106/mL in BO solution (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) containing 10 mM hypotaurine and 4 units/mL heparin. After 18 h of gamete co-culture, presumptive zygotes were cultured in CR1aa (Rosenkrans et al. 1991 Theriogenology 35, 266) supplemented with 5% CS for 8 days at 38.5°C under 5% CO2, 5% O2, 90% N2 atmosphere in air. After in vitro fertilization, Day 7 and Day 8 blastocysts were frozen in 1.5 M ethylene glycol (EG) in Dulbecco's PBS (DPBS) supplemented with 0.1 M sucrose and 20% CS. Embryos were transferred into a freezing medium, loaded into 0.25-mL straws, and allowed to stand for 15–20 min for equilibration. The straws were then plunged into a −7°C methanol bath of a programmable freezer for 1 min, seeded at −7°C, maintained at −7°C for 15 min, cooled to −30°C at the rate of −0.3°C/min, and then plunged into liquid nitrogen. Recipient animals (43 heifers, 131 cows) included those that did not conceive after being artificially inseminated (AI) 3 to 15 times. The frozen–thawed IVF embryos were directly transferred to the recipient animals 7 days after estrus or AI. Pregnancy rates were analyzed by chi-square test. The results are presented in Table 1. There were no significant differences in the pregnancy rates between treatments. However, a slightly higher pregnancy rate was achieved by embryo transfer after AI. These results suggest that embryo transfer may increase the pregnancy rate in repeat-breeder Holstein cattle. Table 1. Pregnancy rates after transfer of IVF frozen–thawed embryos in repeat-breeder Holstein cattle

In recent years, reproductive performance of dairy cows has been declining worldwide, especially among cows of high genetic merit for milk production. The cause of the low fertility may considerably vary across countries and is probably multifactorial. This problem remains unsolved. Further, the first-service conception rate of dairy cows has remarkably decreased worldwide. The number of services required for conception has increased due to the low fertility in dairy cows. However, there are few reports about the relationship between conception rates and the number of services in the current dairy cows. The objective of this study was to investigate whether the number of services affects the conception rates of dairy cows. Data concerning the conception rates was obtained for 8386 Holstein cows from 40 commercial dairy herds in eastern Hokkaido, Japan, from 2006 to 2009. The diagnosis of pregnancy was confirmed by rectal palpation between 30 and 45 days after insemination. The average interval between calving and the first-service was 87.3 days. The average milk yield was 8500 kg. Number of conducting services was from the first to the seventh service. The conception rate was analysed using chi-square test. The results are presented in Table1. The conception rate from the first to the seventh service was 40.2 to 54.0%. The conception rate at the first service was significantly low (40.2%); however, the conception rates did not significantly differ after the second-service. A similar tendency was observed during each year. Moreover, the total percentage of conceptions from the first to the third service was 83.8%. The average number of services per conception was 2.2. The results of this study indicate that the first service yielded the lowest conception rates. The conception rates after the second service did not significantly differ. Moreover, ∼84% of the dairy cows became pregnant between the first service and the third service. However, this result shows that ∼16% of the dairy cows in the herds were repeat breeding. In order to improve fertility, it is necessary to study the factors that affect the first-service conception rates of dairy cows. Moreover, to improve the conception rate of dairy cows, it is important to elucidate the cause of these problems. Table 1.The number and percentage of dairy cows conceiving at each of the services for consecutive services