It is necessary to control the sex ratio (percentage of females) of pre-implantation embryos in the dairy industry. Several studies indicate that, in cattle, varying insemination time does not influence the embryo sex ratio (Rorie et al. 1999 Theriogenology 52, 1273–1280). However, it has been suggested that insemination early in estrus would result in more females, and late in estrus, more males, due to different timing of capacitation and survival of X- and Y-chromosome-bearing spermatozoa in the female reproductive tract (Martinez et al. 2004 Theriogenology 62, 1264–1270). The objective of the present study was to examine whether the time of insemination affects the sex of 7-day embryos in superstimulated Holstein heifers. Donor heifers (14 to 18 months of age, n = 38) were given decreasing doses of FSH IM (total of 28 Armour units, Antrin R-10; Kawasaki-Mitaka, Kanagawa, Japan) twice daily for 4 days between Days 8 and 10 of the estrous cycle. Five (25 mg) and 3 (15 mg) mL of dinoprost (PGF; Veterinary Pronalgon; Pfizer Animal Health, Tokyo, Japan) were administered IM with the seventh and eighth FSH treatments, respectively. The heifers received 50 �g of fertirelin acetate IM (GnRH; Conceral; Schering-Plough Animal Health KK, Tokyo, Japan) 48 h after the first PGF treatment. Heifers were inseminated with 2 straws of frozen–thawed semen into the uterine body at 48 h (n = 18), 60 h (n = 10), or 60 + 72 h (n = 10; control) after the first PGF treatment. In this study, frozen–thawed semen from several bulls was used. Seven-day embryos were recovered by uterine flushing. Grade 1 embryos (IETS classification) were selected for male and female separation using the loop-mediated isothermal amplification procedure. The data (mean � SEM) were compared with Fisher's protected least significant difference test following ANOVA. The mean number of ova/embryos recovered was 12.1 � 1.9 (48 h), 12.8 � 2.8 (60 h), and 11.0 � 1.6 (60 + 72 h). There were no significant differences in the percentages of unfertilized ova (48 h: 6.9 � 2.1%, 15/218; 60 h: 7.8 � 3.2%, 17/128; and 60 + 72 h: 10.0 � 3.6%, 11/111; P > 0.05) and in grade 1 embryos (48 h: 61.2 � 4.9%, 124/218; 60 h: 64.9 � 7.9%, 65/128; and 60 + 72 h: 59.1 � 4.8%, 66/111; P > 0.05) among groups. Furthermore, the female ratio of grade 1 embryos was not significantly different for the 3 insemination times (48 h: 50.0 � 5.8%, 56/124; 60 h: 48.4 � 5.0%, 33/65; and 60 + 72 h: 52.2 � 8.0%, 36/66; P > 0.05). These results indicate that a single early insemination during estrus does not reduce the fertilization rate and embryo quality in superstimulated Holstein heifers. Also, the time of insemination did not appear to have any effect on the sex ratio of embryos from these heifers.
EXPERIMENTAL ANALYSIS OF A PATERNALLY INHERITED EXTRACHROMOSOMAL FACTOR
