Flow cytometric sexing of X- and Y-chromosome-bearing sperm in Sika deer (Cervus nippon)

The objectives of the study were to determine a practical method of using predetermined sexed semen in Sika deer (Cervus nippon). Semen was collected by electro-ejaculation from two Sika stags and transported to the laboratory and separated into X- and Y-chromosome-bearing sperm after analysis and re-analysis (using a modified high-speed cell sorter), or control (unsorted) semen. Eighty-four Sika hinds were inseminated with 2.8 × 107 unsorted (control) or 2.3 × 106 sorted (X or Y) frozen-thawed semen via intra-uterine laparoscopy 58–66 h after removal of intra-vaginal progesterone-impregnated CIDR devices and the administration of 330 IU PMSG at the time of CIDR removal. No significant differences in the post-thaw motility of control (43.4 ± 4.4%), X- (45.3 ± 4.5%) and Y-sorted (43.5 ± 3.2%) samples were recorded. The sorted frozen-thawed sperm (X, 72.5 ± 6.4%: Y, 75.2 ± 5.5%) recorded significantly (P < 0.05) more intact acrosomes following thawing than the unsorted frozen-thawed (68.2 ± 10.2%) sperm. The individual Sika stags had no effect on the post-thaw sperm motility. Sorted frozen-thawed sperm demonstrated a significantly shorter survival time after thawing than the control sperm (P < 0.05). The number of Sika hinds pregnant following insemination with unsorted or control thawed sperm was significantly higher (33/42; 78.6%) than for hinds inseminated with either X- (5/11; 45.5%) or Y-sorted sperm (15/31; 48.4%). Ultimately 14 out of the 15 calves produced by Sika hinds inseminated with Y-sorted sperm were male (92.9%) and 5/5 calves (100%) from Sika hinds inseminated with X-sorted sperm were female. The sex ratio of the calves born to hinds inseminated with sex-sorted sperm significantly (P < 0.05) deviated for the 48.5% (female, 16/33) and 51.5% (male, 17/33) in the control group. All calves were born between 230 d and 243 d of gestation. Male and female calves in the control group had similar birth and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal calves of the predicted sex may be produced after intra-uterine insemination conducted by laparoscopy with low numbers of sex-sorted cryopreserved Sika sperm.