102 Age-related differences in seminal parameters and expression of fertility marker genes in Marwari stallion

The objective of the present study was to observe age-related differences in seminal parameters and expression of fertility marker genes in Marwari stallions. Semen was collected from 9 Marwari stallions (6 ejaculates per stallion) of 3 different age groups (2 to 4 years, GI; 4 to 6 years, GII; and \u003e6 years, GIII) twice a week using an artificial vagina by allowing the stallions to mount a mare. The collected ejaculate was divided into 2 parts. Part A was used for evaluation of various seminal parameters (colour, consistency, total volume, gel volume, gel-free volume, pH, progressive sperm motility, sperm concentration, sperm viability, total sperm morphological abnormalities, plasma membrane integrity, acrosomal integrity, and DNA integrity). Part B was centrifuged to obtain the sperm pellet for DNA extraction. Six fertility-related marker genes (SPATA1, SP17, PLCz, CRISP3, UBB, and PRM1) were examined in the spermatozoa DNA using PCR. Expression levels of these genes were also studied using quantitative real-time PCR. Data obtained were analysed statistically by one-way or two-way analysis of variance using the SPSS computer program (version 20.0; IBM Corp.). No significant difference was observed among the GI, GII, and GIII stallions for seminal parameters, except mean sperm concentration, which was lowest in 2- to 4-year-old stallions (146.06 ± 11.50 million mL−1), intermediate in stallions \u003e6 years old (182.03 ± 8.51 million mL−1), and highest in stallions of 4 to 6 years (270.92 ± 9.12 million mL−1; P \u003c 0.01). The study demonstrated that all six fertility-related genes showed differential expression in stallions from the GI, GII, and GIII groups. In addition, expression levels of the same genes varied across individuals. Expression of SPATA1, SP17, PLCz, CRISP3, UBB, and PRM1 genes was reduced in stallions below 4 years of age compared with older stallions. This result suggests that expression of these genes increases with age, although possibly only up to a certain age. We inferred that stallions around 4–6 years of age can be considered optimum to use for breeding purposes, provided seminal parameters are normal.