Effects of Diluent pH on Enrichment and Performance of Dairy Goat X/Y Sperm

Abstract

In this paper, on the basis of the differences in the hydrogen ion concentration (pH) of the diluent dairy goat semen on X/Y sperm motility, an X/Y sperm enrichment study was conducted to establish a simple and effective method for gender control in dairy goats. Dairy goat semen was diluted using different pH dilutions and was incubated. Then, the X/Y sperm ratio in the isolated upper sperm was determined using the double TaqMan qPCR method. The internal pH change pattern of sperm cells at different pH dilutions was measured using BCECF-AM probe, and the functional parameters of the isolated sperm were tested with the corresponding kit. Next, an in vitro fertilization test was conducted using isolated spermatozoa and oocytes to determine their fertilization rates, the percentages of female embryos, and the expression of genes related to developing potentially fertilized embryos. Results showed that the percentages of the X sperm cells in the upper sperm layer were 67.24% ± 2.61% at sperm dilution pH of 6.2 and 30.45% ± 1.03% at sperm dilution pH of 7.4, which was significantly different from 52.35% ± 1.72% of the control group (pH 6.8) (P < 0.01). Results also showed that there is a relationship between the external pHo and internal pHi of sperm cells. Furthermore, the percentages of female embryos after the in vitro fertilization of the isolated upper sperm with mature oocytes at pH 6.2 and 7.4 were 66.67% ± 0.05 and 29.73% ± 0.04%, respectively, compared with 48.57% ± 0.02% in the control group (pH 6.8). Highly significant differences occurred between groups (P < 0.01). Additionally, no significant difference was observed during the expression of genes related to embryonic development between the blastocysts formed from sperm isolated by changing the pH of the diluent and the control sperm (P > 0.05). Therefore, this study successfully established a simple and effective method for enriched X/Y sperms from dairy goats, which is important for regulating the desired sex progeny during dairy goat breeding and for guiding dairy goat production.

Alkaline Dilution Alters Sperm Motility in Dairy Goat by Affecting sAC/cAMP/PKA Pathway Activity

“…The proportion of intact and uncapacitated sperm in the upper and lower layers was not significantly different from the control group after incubation at pH 7.4 for 40 min. This is consistent with the results of previous studies that used enriched sperm for in vitro fertilization and found no difference in the fertilization rate between the test and the control groups [ 11 ]. A schematic diagram of the mechanism of the effect of the pH of the dilution solution on sperm motility is shown in Figure 10 .…”

“…We established in a previous study [ 11 ] ( Figure 1 A) that the pHi of the sperm in the upper and lower layers of a test tube increased to 6.9 (initial 5.01) over 10 min and then remained unchanged when incubated in a diluent of pH 7.4 ( Figure 1 B,C). The total motility and forward motility of spermatozoa in the upper layer of the tube were found to be significantly higher than that of spermatozoa in the lower layer ( Figure 1 D,E).…”

“…The ratio of X/Y-chromosome-bearing sperm has also been reported to change after incubation of human sperm at different pHs [ 7 , 8 ]. A previous study in our laboratory revealed that, when dairy goat sperm were incubated in alkaline diluent, X-chromosome-bearing sperm could be effectively enriched in the lower layer of the test tube [ 11 ]. However, we did not study the specific mechanism in this previous study.…”

“…After incubation under different conditions, the upper and lower spermatozoa genomic DNA in the test tube were extracted, and the X-chromosome-bearing sperm count and ratio were calculated using the double TaqMan qPCR method (Bio-rad, Hercules, CA, USA) [ 11 ].…”

“…X-chromosome-bearing sperm and Y-chromosome-bearing sperm exhibit different motility activities under different pH conditions [ 9 , 10 ], affecting the sex ratio of offspring. In our previous study, we found that dairy goat sperm incubated in alkaline diluent was enriched in X-chromosome-bearing sperm and that the quality and performance of the enriched X-chromosome-bearing sperm were not affected by the alkaline pH; consequently, the number of female embryos and female kids was significantly increased [ 11 ].…”